Compositions and methods for targeted rna delivery

ABSTRACT

Provided herein are compositions, methods of making the same, and methods for targeted delivery of therapeutic agents for modifying expression and function of target genes, e.g. proteins involved in lipid and cholesterol metabolism such as PCSK9. Further provided herein are compositions and methods of treating conditions related to coronary disease.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. patent applicationSer. No. 17/192,709, filed Mar. 4, 2021, which claims the benefit ofU.S. Provisional Application No. 63/078,982 filed on Sep. 16, 2020 andU.S. Provisional Application No. 62/984,866 filed on Mar. 4, 2020, whichare hereby incorporated by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 23, 2021, isnamed 53989_706_401_SL.txt and is 67,279 bytes in size.

FIELD OF THE DISCLOSURE

The instant disclosure relates to compositions and methods for targeteddelivery of therapeutic agents such as CRISPR-guide RNA and othernucleic acid agents.

BACKGROUND

All publications herein are incorporated by reference to the same extentas if each individual publication or patent application was specificallyand individually indicated to be incorporated by reference. Thefollowing description includes information that may be useful inunderstanding the present disclosure. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

SUMMARY

In one aspect, described herein is a receptor targeting conjugate,comprising a compound of Formula (V):

-   -   wherein,    -   a plurality of the A groups collectively comprise a receptor        targeting ligand;    -   each L¹, L², L³, L⁴, L⁵, L⁶, L⁷, L⁸, L⁹, L¹⁰ and L¹² is        independently substituted or unsubstituted C₁-C₁₂ alkylene,        substituted or unsubstituted C₁-C₁₂ heteroalkylene, substituted        or unsubstituted C₂-C₁₂ alkenylene, substituted or unsubstituted        C₂-C₁₂ alkynylene, —(CH₂CH₂O)_(m)—, —(OCH₂CH₂)_(m)—, —O—, —S—,        —S(═O)—, —S(═O)₂—, —S(═O)(═NR¹)—, —C(═O)—, —C(═N—OR¹)—,        —C(═O)O—, —OC(═O)—, —C(═O)C(═O)—, —C(═O)NR¹—, —NR¹C(═O)—,        —OC(═O)NR¹—, —NR¹C(═O)O—, —NR¹C(═O)NR¹—, —C(═O)NR¹C(═O)—,        —S(═O)₂NR¹—, —NR¹S(═O)₂—, —NR¹—, or —N(OR¹)—;    -   L¹¹ is substituted or unsubstituted —(CH₂CH₂O)_(n)—, or        substituted or unsubstituted —(OCH₂CH₂)_(n)—;    -   each R¹ is independently H or substituted or unsubstituted        C₁-C₆alkyl;    -   R is a lipid, nucleic acid, amino acid, protein, or lipid        nanoparticle;    -   m is an integer selected from 1 to 10; and    -   n is an integer selected from 1 to 200.

In some embodiments, each L¹, L⁴, and L⁷ is independently substituted orunsubstituted C₁-C₁₂ alkylene. In some embodiments, each L¹, L⁴, and L⁷is independently substituted or unsubstituted C₂-C₆ alkylene. In someembodiments, each L¹, L⁴, and L⁷ is C₄ alkylene. In some embodiments,each L², L⁵, and L⁸ is independently —C(═O)NR¹—, —NR¹C(═O)—,—OC(═O)NR¹—, —NR¹C(═O)O—, —NR¹C(═O)NR¹—, or —C(═O)NR¹C(═O)—. In someembodiments, each L², L⁵, and L⁸ is independently —C(═O)NR¹— or—NR¹C(═O)—. In some embodiments, each L², L⁵, and L⁸ is —C(═O)NH—. Insome embodiments, each L³, L⁶, and L⁹ is independently substituted orunsubstituted C₁-C₁₂ alkylene. In some embodiments, each L³ issubstituted or unsubstituted C₂-C₆ alkylene. In some embodiments, L³ isC₄ alkylene. In some embodiments, each L⁶ and L⁹ is independentlysubstituted or unsubstituted C₂-C₁₀ alkylene. In some embodiments, eachL⁶ and L⁹ is independently substituted or unsubstituted C₂-C₆ alkylene.In some embodiments, each L⁶ and L⁹ is C₃ alkylene. In one aspect,described herein is a receptor targeting conjugate, comprising acompound of Formula (VI):

-   -   wherein,    -   a plurality of the A groups collectively comprise a receptor        targeting ligand;    -   each L¹, L², L³, L⁴, L⁵, L⁶, L⁷, L⁸, L⁹, L¹⁰ and L¹² is        independently substituted or unsubstituted C₁-C₁₂ alkylene,        substituted or unsubstituted C₁-C₁₂ heteroalkylene, substituted        or unsubstituted C₂-C₁₂ alkenylene, substituted or unsubstituted        C₂-C₁₂ alkynylene, —(CH₂CH₂O)_(m)—, —(OCH₂CH₂)_(m)—, —O—, —S—,        —S(═O)—, —S(═O)₂—, —S(═O)(═NR¹)—, —C(═O)—, —C(═N—OR¹)—,        —C(═O)O—, —OC(═O)—, —C(═O)C(═O)—, —C(═O)NR¹—, —NR¹C(═O)—,        —OC(═O)NR¹—, —NR¹C(═O)O—, —NR¹C(═O)NR¹—, —C(═O)NR¹C(═O)—,        —S(═O)₂NR¹—, —NR¹S(═O)₂—, —NR¹—, or —N(OR¹)—;    -   L¹¹ is substituted or unsubstituted —(CH₂CH₂O)_(n)—, or        substituted or unsubstituted —(OCH₂CH₂)_(n)—;    -   each R¹ is independently H or substituted or unsubstituted        C₁-C₆alkyl;    -   R is a lipid, nucleic acid, amino acid, protein, or lipid        nanoparticle;    -   m is an integer selected from 1 to 10; and    -   n is an integer selected from 1 to 200.

In some embodiments, each L¹, L⁴, and L⁷ is independently substituted orunsubstituted C₁-C₁₂ alkylene or substituted or unsubstituted C₁-C₁₂heteroalkylene. In some embodiments, each L¹, L⁴, and L⁷ isindependently substituted or unsubstituted C₁-C₁₂ heteroalkylene. Insome embodiments, each L¹, L⁴, and L⁷ is independently substituted orunsubstituted C₁-C₁₂ heteroalkylene comprising 1-10 O atoms. In someembodiments, each L¹, L⁴, and L⁷ is independently—(CH₂CH₂O)_(p1)—(CH₂)_(q1)—; wherein p1 is 1-8; and q1 is 1-6. In someembodiments, each L¹, L⁴, and L⁷ is —(CH₂CH₂O)₃—(CH₂)₂—. In someembodiments, each L¹, L⁴, and L⁷ is independently substituted orunsubstituted C₁-C₁₂ alkylene. In some embodiments, each L¹, L⁴, and L⁷is independently substituted or unsubstituted C₂-C₆ alkylene. In someembodiments, each L¹, L⁴, and L⁷ is C₄ alkylene. In some embodiments,each L², L⁵, and L⁸ is independently —C(═O)NR¹—, —NR¹C(═O)—,—OC(═O)NR¹—, —NR¹C(═O)O—, —NR¹C(═O)NR¹—, or —C(═O)NR¹C(═O)—. In someembodiments, each L², L⁵, and L⁸ is independently —C(═O)NR¹— or—NR¹C(═O)—. In some embodiments, each L², L⁵, and L⁸ is —NHC(═O)—. Insome embodiments, each L², L⁵, and L⁸ is —C(═O)NH—. In some embodiments,each L³, L⁶, and L⁹ is independently substituted or unsubstituted C₁-C₁₂heteroalkylene. In some embodiments, each L³, L⁶, and L⁹ isindependently substituted or unsubstituted C₁-C₁₂ heteroalkylenecomprising 1-10 O atoms. In some embodiments, each L³, L⁶, and L⁹ isindependently —(CH₂CH₂O)_(p2)—(CH₂CH₂CH₂O)_(q2)—; wherein p2 is 1-8; andq2 is 1-6. In some embodiments, each L³, L⁶, and L⁹ is—(CH₂CH₂O)—(CH₂CH₂CH₂O)—. In some embodiments, each L³, L⁶, and L⁹ isindependently —(CH₂CH₂CH₂O)_(q3)—; wherein q3 is 1-8. In someembodiments, each L³, L⁶, and L⁹ is —(CH₂CH₂CH₂O)₂—. In someembodiments, L¹⁰ is substituted or unsubstituted C₁-C₁₂ alkylene. Insome embodiments, L¹⁰ is substituted or unsubstituted C₁-C₄ alkylene. Insome embodiments, L¹⁰ is C₂ alkylene. In some embodiments, L¹¹ is—(OCH₂CH₂)_(n)—. In some embodiments, n is 1-100. In some embodiments, nis 2-50. In some embodiments, n is 2, 12, 37, or 45. In someembodiments, L¹² is —O—, —C(═O)O—, —C(═O)NR¹—, —NR¹C(═O)—, or—NR¹C(═O)O—. In some embodiments, L¹² is —C(═O)O— or —NR¹C(═O)O—. Insome embodiments, L¹² is —C(═O)O—. In some embodiments, L¹² is—NHC(═O)O—. In some embodiments, L¹² is —NHC(═O)—. In some embodiments,A binds to a lectin. In some embodiments, the lectin is anasialoglycoprotein receptor (ASGPR). In some embodiments, A isN-acetylgalactosamine (GalNAc) or a derivative

or a derivative thereof. A is N-acetylgalactosamine (GalNAc)

or a derivative thereof. In some embodiments, each R¹ is independently Hor —CH₃. In some embodiments, each R¹ is H. In some embodiments, the Rcomprises one or more of fatty alcohols, fatty acids, glycerolipids,glycerophospholipids, sphingolipids, saccharolipids, polyketides, sterollipids, and prenol lipids. In some embodiments, the R comprises one ormore fatty alcohols. In some embodiments, each fatty alcohol isindependently a saturated, monounsaturated, or polyunsaturated fattyalcohol. In some embodiments, the fatty alcohol comprises one or more aC₂-C₂₆ fatty alcohol. In some embodiments, the fatty alcohol comprisestwo or more a C₂-C₂₆ fatty alcohol. In some embodiments, each fattyalcohol is a C12, C14, C16, C18, C20, or C22 fatty alcohol. In someembodiments, each fatty alcohol is independently docosahexaenol,eicosapentaenol, oleyl alcohol, stearyl alcohol,(9Z,12Z)-octadeca-9,12-dien-1-yl alcohol, (Z)-docos-13-en-1-yl alcohol,docosanyl alcohol, (E)-octadec-9-en-1-yl alcohol, icosanyl alcohol,(9Z,12Z,15Z)-octadeca-9,12,15-trien-1-yl alcohol, or palmityl alcohol.In some embodiments, each fatty alcohol is a stearyl alcohol. In someembodiments, the R comprises one or more sterol lipids. In someembodiments, the R comprises one or more of vitamins. In someembodiments, each vitamin is independently a vitamin A, vitamin D,vitamin E, or vitamin K. In some embodiments, the R comprises a nucleicacid. In some embodiments, the nucleic acid is a mRNA, guide RNA, siRNA,antisense oligonucleotide, aptamer, microRNA, immunostimulatoryoligonucleotide, splice switching oligonucleotide, self-amplifying RNA,circular RNA or DNA, but not limited to the aforementioned. In someembodiments, the R comprises a protein. In some embodiments, the proteinis a gene editor protein, an antibody, an antigen-binding antibodyfragment or a peptide, but not limited to the aforementioned.

In one aspect, described herein is a receptor targeting conjugate,comprising a compound from Table 4.

In one aspect, described herein is a nanoparticle compositioncomprising: (a) one or more nucleic acid molecular entities; and (b) areceptor targeting conjugate as described herein. In some embodiments,the receptor targeting conjugate comprises from about 0.001 mol % toabout 20 mol % of the total lipid content present in the nanoparticlecomposition. In some embodiments, the receptor targeting conjugatecomprises from about 0.01 mol % to about 1 mol % of the total lipidcontent present in the nanoparticle composition. In some embodiments,comprising a sterol or a derivative thereof, comprising from 10 mol % to70 mol % of the total lipid content present in the nanoparticlecomposition. In some embodiments, the sterol or the derivative thereofis cholesterol or a cholesterol derivative. In some embodiments, thecholesterol or the cholesterol derivative comprises from 20 mol % to 50mol % of the total lipid content present in the nanoparticlecomposition. In some embodiments, the nanoparticle composition comprisesa phospholipid, comprising from 1 mol % to 20 mol % of the total lipidcontent present in the nanoparticle composition. In some embodiments,the phospholipid comprises from about 5 mol % to about 15 mol % of thetotal lipid content present in said nanoparticle composition. In someembodiments, the phospholipid is selected from1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC),2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC),1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and sphingomyelin.In some embodiments, the phospholipid is DSPC. In some embodiments, thenanoparticle composition comprises a stealth lipid, comprising from 0.1mol % to 6 mol % of the total lipid content present in the nanoparticlecomposition. In some embodiments, the stealth lipid comprises about 2.0mol % to about 2.5 mol % of the total lipid content present in saidnanoparticle composition. In some embodiments, the stealth lipid is aPEG-lipid that has a number average molecular weight of from about 200Da to about 5000 Da. In some embodiments, the nanoparticle compositioncomprises an amino lipid, comprising from about 10 mol % to about 60 mol% of the total lipid content present in the nanoparticle composition. Insome embodiments, the nanoparticle composition comprises an antioxidant.In some embodiments, the antioxidant comprisesethylenediaminetetraacetic acid (EDTA). In some embodiments, the one ormore nucleic acid molecular entities comprise a single guide RNA (sgRNA)or guide RNA (gRNA) targeting a disease causing gene of interestproduced in the hepatocytes. In some embodiments, the one or morenucleic acid molecular entities comprise an mRNA that encodes a Casnuclease. In some embodiments, at least one of the one or more nucleicacid molecular entities comprises a chemical modification. In someembodiments, the chemical modification is a 2′-F modification, aphosphorothioate internucleotide linkage modification, acyclicnucleotides, LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl,2′-C-allyl, 2′-deoxy, 2′-fluoro, 2′-O—N-methylacetamido (2′-O-NMA), a2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE), 2′-O-aminopropyl (2′-O-AP),4′-O-methyl, or a 2′-ara-F modification. In some embodiments, thechemical modification is a 2′-O-methyl modification.

In one aspect, provided herein is a pharmaceutical compositioncomprising a herein described receptor targeting conjugate or a hereindescribed nanoparticle composition, and an excipient or carrier. In someembodiments, the pharmaceutical composition comprises an mRNA encoding agene editor nuclease. In some embodiments, the pharmaceuticalcomposition comprises one or more guide RNA molecules. In someembodiments, the pharmaceutical composition comprises two or more guideRNA molecules. In some embodiments, the two or more guide RNA moleculestarget two or more genes of interest. In some embodiments, the mRNAencodes Cas9 nuclease. In some embodiments, the mRNA encodes a baseeditor nuclease. In some embodiments, the mRNA and the one or more guideRNA molecules are present in the same nanoparticle composition. In someembodiments, the mRNA and the one or more guide RNA molecules arepresent in different nanoparticle compositions. In some embodiments, aratio of the gRNA molecules to the mRNA in the pharmaceuticalcomposition is from about 0.01 to about 100 by weight or by mole. Insome embodiments, a ratio of said gRNA molecules to said mRNA in saidpharmaceutical composition is about 50:1, about 40:1, about 30:1, about20:1, about 18:1, about 16:1, about 14:1, about 12:1, about 10:1, about9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1,about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about1:6, about 1:7, about 1:8, about 1:9, or about 1:10 by weight or bymole.

In one aspect, provided herein is a pharmaceutical compositioncomprising: (a) a first receptor targeting conjugate or a firstnanoparticle composition as described herein, and (b) a second receptortargeting conjugate or a second nanoparticle composition as describedherein. In some embodiments, the first nanoparticle compositioncomprises a gene editor mRNA. In some embodiments, the secondnanoparticle composition comprises one or more guide RNA molecules. Insome embodiments, a ratio of guide RNA molecules to mRNA in saidpharmaceutical composition is about 50:1, about 40:1, about 30:1, about20:1, about 18:1, about 16:1, about 14:1, about 12:1, about 10:1, about9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1,about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about1:6, about 1:7, about 1:8, about 1:9, or about 1:10 by weight or bymole.

In one aspect, provided herein is a method of delivering a nucleic acidto a cell, the method comprising contacting the cell with a nanoparticlecomposition or a pharmaceutical composition as described herein, wherebythe nucleic acid is delivered to said cell. In some embodiments, thecell is contacted in vivo, ex vivo, or in intro.

In one aspect, provided herein is a method of producing a polypeptide ofinterest in a cell, the method comprising contacting said cell with ananoparticle composition or a pharmaceutical composition as describedherein, whereby the nucleic acid is capable of being translated in saidcell to produce the polypeptide.

In one aspect, provided herein is a method of treating a disease orcondition in a subject, the method comprising administering to thesubject a therapeutically effective amount of a pharmaceuticalcomposition as described herein. In some embodiments, the disease orcondition is a coronary disease. In some embodiments, the subject islow-density lipoprotein receptor (LDLR)-deficient. In some embodiments,the subject has heterozygous familial hypercholesterolemia (HeFH),homozygous familial hypercholesterolemia (HoFH) or clinicalatherosclerotic cardiovascular disease (ASCVD). In some embodiments, thesubject is at high risk of cardiovascular events. In some embodiments,the subject requires additional lowering of low-density lipoproteincholesterol (LDL-C) despite maximally tolerated lipid-lowering therapy.

In one aspect, provided herein is a method of delivering a nucleic acidmolecular entity to the liver of a subject, comprising administering tothe subject a pharmaceutical composition as described herein, therebydelivering the nucleic acid molecular entity.

In one aspect, described herein is a nucleotide conjugate comprising:(a) a nucleic acid, and (b) a targeting moiety connected to the nucleicacid in (a), wherein the targeting moiety comprises a structure ofTable 1. In some embodiments, the targeting moiety further comprises acoupling sequence that hybridizes with the nucleic acid in (a). In oneaspect, described herein is a nucleotide conjugate comprising: (a) anucleic acid, and (b) a targeting moiety connected to the nucleic acidin (a), wherein the targeting moiety comprises a coupling sequence thathybridizes with the nucleic acid in (a). In some embodiments, thenucleic acid comprises a single stranded, double stranded, a partiallydouble stranded, or a hairpin stem-loop nucleic acid, and wherein thetargeting moiety is a receptor targeting moiety. In some embodiments,the targeting moiety binds to a lectin. In some embodiments, the lectinis an asialoglycoprotein receptor (ASGPR). In some embodiments, thetargeting moiety comprises one or more N-acetylgalactosamine (GalNAc) orGalNAc derivatives. In some embodiments, the targeting moiety comprisesone or more N-acetylgalactosamine (GalNAc) or GalNAc derivatives and aspacer. In some embodiments, the targeting moiety comprises one or moregalactose or galactose derivatives. In some embodiments, the targetingmoiety comprises one or more galactose or galactose derivatives and aspacer. In some embodiments, the spacer comprises polyethylene glycol,substituted or unsubstituted C₁-C₁₂ alkylene, or both, wherein thepolyethylene glycol has from 1 to 5 repeating units. In someembodiments, the targeting moiety is linked to one or more strands ofthe nucleic acid through one or more linkers. In some embodiments, thetargeting moiety comprises a structure of Table 1. In some embodiments,the coupling sequence hybridizes with the nucleic acid in (a). In someembodiments, the coupling sequence hybridizes with an extension in thenucleic acid in (a). In some embodiments, the targeting moiety isattached to the 5′ end of the nucleic acid sequence, the 3′ end of thenucleic acid sequence, or the middle of the nucleic acid sequence. Insome embodiments, the targeting moiety comprises at least two GalNAcs orGalNAc derivatives. In some embodiments, the targeting moiety comprisesat least three GalNAcs or GalNAc derivatives. In some embodiments, theGalNAcs or GalNAc derivatives are connected to the nucleic acid in (a)via a linker in the targeting moiety, via hybridization of the couplingsequence in the targeting moiety that hybridizes with the nucleic acidin (a), or via a combination thereof. In some embodiments, the targetingmoiety comprises at least two galactose or galactose derivatives. Insome embodiments, the targeting moiety comprises at least threegalactoses or galactose derivatives. In some embodiments, the galactosesor galactose derivatives are connected to the nucleic acid in (a) via alinker in the targeting moiety, via hybridization of the couplingsequence in the targeting moiety that hybridizes with the nucleic acidin (a), or via a combination thereof. In some embodiments, the targetingmoiety comprises at least two coupling sequences that hybridize with thenucleic acid in (a). In some embodiments, the at least two couplingsequences are identical. In some embodiments, the at least two couplingsequences are different. In some embodiments, the nucleotide conjugatefurther comprises a second targeting moiety. In some embodiments, thesecond targeting moiety binds to an asialoglycoprotein receptor (ASGPR).In some embodiments, the second targeting moiety is linked to one ormore strands the nucleic acid through a spacer and/or through one ormore linkers. In some embodiments, the second targeting moiety comprisesa GalNAc or GalNAc derivative. In some embodiments, the second targetingmoiety comprises at least three GalNAc moieties or GalNAc derivatives.In some embodiments, the GalNAc or GalNAc derivatives are connected tothe nucleic acid in (a) via a linker in the targeting moiety, viahybridization of the coupling sequence in the targeting moiety thathybridizes with the nucleic acid in (a), or via a combination thereof.In some embodiments, the second targeting moiety comprises a galactoseor galactose derivative. In some embodiments, the second targetingmoiety comprises at least three galactose moieties or galactosederivatives. In some embodiments, the galactose or galactose derivativesare connected to the nucleic acid in (a) via a linker in the targetingmoiety, via hybridization of the coupling sequence in the targetingmoiety that hybridizes with the nucleic acid in (a), or via acombination thereof. In some embodiments, the second targeting moietycomprises a structure of Table 1. In some embodiments, the secondtargeting moiety comprises a coupling sequence that hybridizes with thenucleic acid. In some embodiments, the second targeting moiety isattached to the 5′ end of the nucleic acid, the 3′ end of the nucleicacid, or the middle of the nucleic acid. In some embodiments, thenucleic acid in (a) comprises RNA or DNA. In some embodiments, thecoupling sequence comprises RNA, DNA, chemically modified RNA,chemically modified DNA, or a hybrid of DNA and RNA. In someembodiments, the coupling sequence comprises one or more of (a), (c),(g), (u), (A)n, (T)n, (U)n, (a)n, or (u)n, wherein n is an integer noless than 3, wherein a is 2′-O-methyladenosine (2′-OMe A), wherein c is2′-O-methylacytidine (2′-OMe-C), wherein g is 2′-O-methylacytidineguanine (2′-OMe-G), and wherein u is 2′-O-methyluridine (2′-OMe-U). Insome embodiments, the coupling sequence comprises one or more of (a),(c), (g), (u), (A)n, (T)n, (U)n, (a)n, or (u)n, wherein n is an integerno less than 3, wherein a is 2′-O-methyladenosine (2′-OMe A), wherein cis 2′-O-methylacytidine (2′-OMe-C), wherein g is 2′-O-methylacytidineguanine (2′-OMe-G), and wherein u is 2′-O-methyluridine (2′-OMe-U). Insome embodiments, the (a), (c), (g), or (u) is scattered along thenucleic acid or the coupling sequence. In some embodiments, the nucleicacid and the coupling sequence comprise one or more G-C base pairingwithin a hybridization duplex wherein the coupling sequence hybridizeswith the nucleic acid and wherein said one or more G-C base pairingincreases stability of the hybridization duplex. In some embodiments,the linker comprises a covalent linker. In some embodiments, the linkercomprises a non-covalent linker. In some embodiments, the linkercomprises a monovalent linker, a bivalent linker, a trivalent linker, ora combination thereof. In some embodiments, the linker comprises abiocleavable linker. In some embodiments, the linker comprises anon-biocleavable linker. In some embodiments, the linker comprises aphosphate, phosphorothioate, amide, ether, oxime, hydrazine orcarbamate. In some embodiments, the linker is a phosphate orphosphorothioate. In some embodiments, the nucleic acid in (a) comprisesa chemical modification. In some embodiments, the nucleic acid in (a)comprises a 2′-F modification, a phosphorothioate internucleotidelinkage modification, acyclic nucleotides, LNA, HNA, CeNA,2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy,2′-fluoro, 2′-O—N-methylacetamido (2′-O-NMA), a2′-O-dimethylaminoethoxyethyl (2′-0-DMAEOE), 2′-0-aminopropyl (2′-O-AP),4′-O-methyl, or a 2′-ara-F modification. In some embodiments, thenucleic acid comprises a 2′-O-methyl modification. In some embodiments,the nucleic acid comprises a phosphorothioate internucleotide linkagemodification. In some embodiments, the nucleic acid is capable ofhybridizing with a target sequence within a target gene of a genome. Insome embodiments, the nucleic acid comprises a mRNA, siRNA, shRNA,antisense oligonucleotide, microRNA, anti-microRNA or antimir, supermir,antagomir, ribozyme, triplex-forming oligonucleotide, decoyoligonucleotide, splice-switching oligonucleotide, immunostimulatoryoligonucleotide, RNA activator, Ul adaptor, guide RNA, or anycombinations thereof. In some embodiments, the nucleic acid encodes aprotein. In some embodiments, the nucleic acid is a CRISPR enzyme. Insome embodiments, the nucleic acid is a guide RNA capable of forming acomplex with a CRISPR enzyme. In some embodiments, the guide RNA is asingle guide RNA or a dual guide RNA. In some embodiments, the CRISPRenzyme is selected from the group consisting of Cas9, Cpf1, CasX, CasY,C2c1, C2c3, and base editor fusion protein. In some embodiments, thenucleic acid further comprises a mRNA encoding the CRISPR enzyme. Insome embodiments, the CRISPR enzyme results in an alteration in thetarget sequence. In some embodiments, the target gene is involved in alipid metabolism pathway. In some embodiments, the target gene isselected from the group consisting of PCSK9, ANGPTL3, APOC3, LPA, APOB,MTP, ANGPTL4, ANGPTL8, APOA5, APOE, LDLR, IDOL, NPC1L1, ASGR1, TM6SF2,GALNT2, GCKR, LPL, MLXIPL, SORT1, TRIB1, MARC1, ABCG5, and ABCG8. Insome embodiments, the guide RNA comprises a sequence selected fromsequences of Table 3. In some embodiments, the guide RNA comprises asequence selected from sequences of Table 5.

In one aspect, described herein is a particle comprising the describednucleotide conjugate and the described CRISPR enzyme. In someembodiments, the particle is a lipid nanoparticle, a liposome, aninorganic nanoparticle, or an RNP.

In one aspect, described herein is a cell comprising the nucleotideconjugate of any one of the preceding claims. In some embodiments, thecell is a prokaryotic cell, a eukaryotic cell, a vertebrate cell, amouse cell, a non-human primate cell, or a human cell.

In one aspect, described herein is a pharmaceutical compositioncomprising the described nucleotide conjugate, the particle, or thecell. In some embodiments, the pharmaceutical composition comprises apharmaceutically acceptable adjuvant, diluent, carrier, preservative,excipient, buffer, stabilizer, or a combination thereof. In someembodiments, the carrier comprises solvents, dispersion media,dispersion or suspension aids, surface active agents, isotonic agents,thickening or emulsifying agents, preservatives, lipids, lipidoids,polymers, lipoplexes, core-shell nanoparticles, hyaluronidase,nanoparticle mimics, or combinations thereof.

In one aspect, described herein is a kit comprising the describednucleotide conjugate.

In one aspect, described herein is a method for reducing the risk ofcoronary disease in a subject in need thereof, comprising administeringto the subject an effective amount of the described nucleotideconjugate. In one aspect, described herein is a method for reducing therisk of coronary disease in a subject in need thereof, comprisingadministering to the subject an effective amount of a pharmaceuticalcomposition, said pharmaceutical composition comprising (a) a nucleicacid, and (b) a targeting moiety connected to the nucleic acid in (a),wherein the targeting moiety comprises a structure of Table 1. In oneaspect, described herein is a method of delivering a nucleic acid to theliver of a subject, comprising administering to the subject said nucleicacid connected to a targeting moiety, wherein the targeting moietycomprises a structure of Table 1. In some embodiments, the targetingmoiety further comprises a coupling sequence that hybridizes with thenucleic acid in (a). In one aspect, described herein is a method forreducing the risk of coronary disease in a subject in need thereof,comprising administering to the subject an effective amount of apharmaceutical composition, said pharmaceutical composition comprising(a) a nucleic acid, and (b) a targeting moiety connected to the nucleicacid in (a), wherein the targeting moiety comprises a coupling sequencethat hybridizes with the nucleic acid in (a). In one aspect, describedherein is a method of delivering a nucleic acid to the liver of asubject, comprising administering to the subject said nucleic acidconnected to a targeting moiety, wherein the targeting moiety comprisesa coupling sequence that hybridizes with the nucleic acid. In someembodiments, the nucleic acid comprises a single stranded, doublestranded, a partially double stranded, or a hairpin stem-loop nucleicacid, and wherein the targeting moiety is a receptor targeting moiety.In some embodiments, the targeting moiety binds to a lectin. In someembodiments, the lectin is an asialoglycoprotein receptor (ASGPR). Insome embodiments, the targeting moiety comprises one or moreN-acetylgalactosamine (GalNAc) or GalNAc derivatives. In someembodiments, the targeting moiety comprises at least three GalNAc orGalNAc derivatives. In some embodiments, the targeting moiety comprisesone or more galactose or galactose derivatives. In some embodiments, thetargeting moiety comprises at least three galactose or galactosederivatives. In some embodiments, the nucleic acid comprises (i) a guideRNA and a nuclease mRNA or (ii) a guide RNA complexed in a nuclease RNP,and wherein the guide RNA is capable of directing the nuclease to atarget sequence in a target gene. In some embodiments, the guide RNAcomprises a single guide RNA or a dual guide RNA. In some embodiments,the nuclease is a CRISPR enzyme. In some embodiments, the CRISPR enzymeselected from the group consisting of Cas9, Cpf1, CasX, CasY, C2c1,C2c3, and base editor fusion protein. In some embodiments, the CRISPRenzyme results in an alteration in the target sequence. In someembodiments, the administration results in reduced expression of thetarget gene in the liver of the subject. In some embodiments, expressionof the target gene in the liver of the subject is reduced by at least1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%,at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or greater than 99.99% as compared to a control tissue of thesubject. In some embodiments, the target gene is associated with acoronary disease. In some embodiments, the target gene is selected fromthe group consisting of PCSK9, ANGPTL3, APOC3, LPA, APOB, MTP, ANGPTL4,ANGPTL8, APOA5, APOE, LDLR, IDOL, NPC1L1, ASGR1, TM6SF2, GALNT2, GCKR,LPL, MLXIPL, SORT1, TRIB1, MARC1, ABCG5, and ABCG8. In some embodiments,the coupling sequence comprises RNA, DNA, chemically modified RNA,chemically modified DNA, or a hybrid of DNA and RNA. In someembodiments, the coupling sequence comprises (A)n, (T)n, (U)n, (a)n, or(u)n, wherein n is an integer no less than 3, wherein a is2′-O-methyladenosine (2′-OMe A), and wherein u is 2′-O-methyluridine(2′-OMe-U). In some embodiments, the nucleic acid in (a) comprises (A)n,(T)n, (U)n, (a)n, or (u)n, wherein n is an integer no less than 3,wherein a is 2′-O-methyladenosine, and wherein u is 2′-O-methyluridine.In some embodiments, the targeting moiety is linked to the nucleic acidin (a) via a linker in the targeting moiety, via hybridization of thecoupling sequence in the targeting moiety that hybridizes with thenucleic acid in (a), or via a combination thereof. In some embodiments,the linker comprises a covalent linker. In some embodiments, the linkercomprises a phosphate, phosphorothioate, amide, ether, oxime, hydrazineor carbamate. In some embodiments, the linker is a phosphate orphosphorothioate. In some embodiments, the nucleic acid in (a) comprisesa chemical modification. In some embodiments, the nucleic acid in (a)comprises a 2′-F modification, a phosphorothioate internucleotidelinkage modification, acyclic nucleotides, LNA, HNA, CeNA,2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy,2′-fluoro, 2′-O—N-methylacetamido (2′-O-NMA), a2′-O-dimethylaminoethoxyethyl (2′-0-DMAEOE), 2′-0-aminopropyl (2′-O-AP),or a 2′-ara-F modification. In some embodiments, the nucleic acidcomprises a 2′-O-methyl modification. In some embodiments, the nucleicacid comprises a phosphorothioate internucleotide linkage modification.In some embodiments, the level of the nucleic acid in the liver of thesubject is at least 1.5, at least 2, at least 2.5, at least 3, at least5, at least 10, at least 15, at least 20, at least 30, at least 40, atleast 50 folds higher as compared to other tissues of the subject atleast 1 hours, 2 hours, 6 hours, 12 hours, 24 hours, 2 days, 1 week, 2weeks, 3 weeks, 6 weeks, or 8 weeks post delivery. In some embodiments,the effective amount is about 1 mg/kg to about 10 mg/kg. In someembodiments, the administration results in reduced blood triglyceridesand/or reduced low-density lipo-protein cholesterol in the subject inneed thereof. In some embodiments, the administration is performedintravenously, intrathecally, intramuscularly, intraventricularly,intracerebrally, intracerebellarly, intracerebroventricularly,intraperenchymally, subcutaneously, or a combination thereof.

In one aspect, described herein is a method for reducing the risk ofcoronary disease in a subject in need thereof, the method comprisingadministering to the subject a pharmaceutical composition comprising (a)(i) a single guide RNA and a nuclease mRNA, (ii) a dual guide RNA and anuclease mRNA, (iii) a single guide RNA and an RNP, or (iv) a dual guideRNAs and an RNP; and (b) a asialoglycoprotein receptor (ASGPR) targetingmoiety connected to the nucleic acid in (a), wherein the single guideRNA or the dual guide RNA comprises 4 or more 2′-O-methyl modificationsand 2 or more phosphorothioate internucleotide linkages, wherein thetargeting moiety comprises a structure of Table 1, and wherein the guideRNA hybridizes with a PCSK9 gene. In one aspect, described herein is amethod for reducing the risk of coronary disease in a subject in needthereof, the method comprising administering to the subject apharmaceutical composition comprising (a) (i) a single guide RNA and anuclease mRNA, (ii) a dual guide RNA and a nuclease mRNA, (iii) a singleguide RNA and an RNP, or (iv) a dual guide RNAs and an RNP; and (b) atargeting moiety connected to the nucleic acid in (a), wherein thesingle guide RNA or the dual guide RNA comprises 4 or more 2′-O-methylmodifications and two or more phosphorothioate internucleotide linkages,wherein the targeting moiety comprises a coupling sequence thathybridizes with the single guide RNA in (a), and wherein the guide RNAhybridizes with a PCSK9 gene. In some embodiments, the nuclease mRNAand/or the single guide RNA comprises at least one chemicalmodification. In some embodiments, the chemical modification is selectedfrom the group consisting of a 2′-F modification, phosphorothioateinternucleotide linkage modification, acyclic nucleotides, LNA, HNA,CeNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy,2′-fluoro, 2′-O—N-methylacetamido (2′-O-NMA), a2′-O-dimethylaminoethoxyethyl (2′-0-DMAEOE), 2′-0-aminopropyl (2′-O-AP),4′-O-methyl, and a 2′-ara-F modification. In some embodiments,administrating of the nucleic acid conjugate results in a reduced levelof immune response as compared to a control nucleic acid conjugatewithout said chemical modification.

In one aspect, described herein is a nucleotide conjugate comprising astructure of Formula (IV)

wherein each X is independently H or a protecting group, R^(A) is —OX or—NHAc, Y is O or S, and W represents

-   -   (a) (i) a single guide RNA and a nuclease mRNA, (ii) a dual        guide RNA and a nuclease mRNA, (iii) a single guide RNA and an        RNP, or (iv) a dual guide RNAs and an RNP; or    -   (b) a coupling sequence.

In some embodiments, the one or more linkers comprise a structureselected from the group consisting of:

In some embodiments, each of the linkers independently has a structureof -(L¹)_(k1)-(L²)_(k2)-(L³)_(k3)-(L⁴)_(k4)-, wherein each of k1, k2,k3, and k4 is independently 0, 1 or 2, and each of the L¹, L², L³ and L⁴is independently selected from —O—, —S—, S(═O)₁₋₂—, —C(═O)—, —C(═S)—,—NR^(L)—, —OC(═O)—, —C(═O)O—, —OC(═O)O—, —C(═O)NR^(L)—, —OC(═O)NR^(L)—,—NR^(L)C(═O)—, —NR^(L)C(═O) NR^(L)—, —P(═O)R^(L)—,—NR^(L)S(═O)(═NR^(L))—, —NR^(L)S(═O)₂—, —S(═O)₂NR^(L)—, —N═N—,—(CH₂—CH₂—O)₁₋₆—, linear or branched C₁₋₆ alkylene, linear or branchedC₂₋₆ alkenylene, linear or branched C₂₋₆ alkynylene, C₃-C₈cycloalkylene, C₂-C₇ heterocycloalkylene, C₆-C₁₀ arylene, and C₅-C₉heteroarylene, wherein the alkylene, alkenylene, alkynylene,cycloalkylene, cycloalkylene, arylene, or heteroarylene is substitutedor unsubstituted, and wherein each R^(L) is independently H, D, cyano,halogen, substituted or unsubstituted C₁-C₆ alkyl, —CD₃, —OCH₃, —OCD₃,substituted or unsubstituted C₁-C₆ haloalkyl, substituted orunsubstituted C₁-C₆ heteroalkyl, substituted or unsubstitutedC₃-C₈cycloalkyl, substituted or unsubstituted C₂-C₇ heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl. In some embodiments, each R^(L) is independently H,substituted or unsubstituted C₁-C₆ alkyl, —OCH₃, substituted orunsubstituted C₁-C₆ haloalkyl, substituted or unsubstituted C₁-C₆heteroalkyl, substituted or unsubstituted C₃-C₈cycloalkyl, orsubstituted or unsubstituted C₂-C₇ heterocycloalkyl. In someembodiments, the sum of k1, k2, k3, and k4 is 1, 2, or 3.

In one aspect, described herein is a method of preparing a formulationcomprising nanoparticles, wherein the nanoparticles comprise (i) one ormore nucleic acid molecular entities, (ii) one or more lipids selectedfrom a sterol or a derivative thereof, a phospholipid, a stealth lipid,and an amino lipid, and (iii) a receptor targeting conjugate, the methodcomprising: (a) providing a first solution comprising the one or morenucleic acid molecular entities; (b) providing a second solutioncomprising at least one of the one or more lipids; (c) mixing the firstsolution and the second solution, thereby producing a mixture comprisingnanoparticles that comprise the one or more nucleic acid molecularentities and the one or more lipids; (d) mixing the receptor targetingconjugate with the nanoparticles produced in step (c); (e) incubatingthe nanoparticles; and (f) optionally carrying out a buffer exchangeprocess. In some embodiments, the receptor targeting conjugate iscombined with the one or more lipids after the mixing step in (c). Insome embodiments, the receptor targeting conjugate is added in adilution buffer, and wherein the dilution buffer is mixed with preformednucleic acid-lipid nanoparticles coming out of an inline mixing chamberthereby forming the nanoparticles. In some embodiments, the receptortargeting conjugate is introduced after an addition of a dilution bufferto the mixture and holding the diluted mixture for a period of time. Insome embodiments, the holding time is between 1 and 120 minutes. In someembodiments, the holding time is between 1 and 90 minutes, between 1 and60 minutes, or between 10 and 40 minutes. In some embodiments, theholding time is about 30 minutes. In some embodiments, the receptortargeting conjugate is introduced to the nanoparticles after bufferexchange. In some embodiments, the receptor targeting conjugate isintroduced to the nanoparticles after buffer exchange and concentration,but prior to storage. In some embodiments, the receptor targetingconjugate is introduced to the nanoparticles after storage and thawing,and prior to dosing or evaluation. In one aspect, described herein is amethod of preparing a formulation comprising nanoparticles, wherein thenanoparticles comprise (i) one or more nucleic acid molecular entities,(ii) one or more lipids selected from a sterol or a derivative thereof,a phospholipid, a stealth lipid, and an amino lipid, and (iii) areceptor targeting conjugate, the method comprising: (a) providing afirst solution comprising the one or more nucleic acid molecularentities; (b) providing a second solution comprising at least one of theone or more lipids; (c) inline mixing of the first solution and thesecond solution, thereby producing a mixture comprising nanoparticlesthat comprise the one or more nucleic acid molecular entities and theone or more lipids; (d) inline mixing of the receptor targetingconjugate to the mixture of step (c), thereby producing a mixturecomprising nanoparticles that comprise the one or more nucleic acidmolecular entities, the one or more lipids, and the receptor targetingconjugate; (e) diluting the mixture of step (d) by adding a dilutionbuffer; and (f) optionally carrying out a buffer exchange process. Insome embodiments, the inline mixing of step (c) and the inline mixing ofstep (d) are performed successively. In one aspect, described herein isa method of preparing a formulation comprising nanoparticles, whereinthe nanoparticles comprise (i) one or more nucleic acid molecularentities, (ii) one or more lipids selected from a sterol or a derivativethereof, a phospholipid, a stealth lipid, and an amino lipid, and (iii)a receptor targeting conjugate, the method comprising: (a) providing afirst solution comprising the one or more nucleic acid molecularentities; (b) providing a second solution comprising (i) at least one ofthe one or more lipids and (ii) at least a portion of the receptortargeting conjugate; (c) mixing the first solution and the secondsolution, thereby producing a mixture comprising nanoparticles; (d)optionally incubating the nanoparticles; and (e) optionally carrying outa buffer exchange process. In some embodiments, the second solutioncomprises all the receptor targeting conjugate. In one aspect, describedherein is a method of preparing a formulation comprising nanoparticles,wherein the nanoparticles comprise (i) one or more nucleic acidmolecular entities, (ii) one or more lipids selected from a sterol or aderivative thereof, a phospholipid, a stealth lipid, and an amino lipid,and (iii) a receptor targeting conjugate, the method comprising: (a)providing a first solution comprising the one or more nucleic acidmolecular entities; (b) providing a second solution comprising at leastone of the one or more lipids; (c) mixing the first solution and thesecond solution, thereby producing a mixture comprising nanoparticlesthat comprise the one or more nucleic acid molecular entities and theone or more lipids; (d) combining the receptor targeting conjugate withthe one or more lipids, wherein at least a portion of the receptortargeting conjugate is combined with the one or more lipids prior to orconcurrently with the mixing step; (e) optionally incubating thenanoparticles; and (f) optionally carrying out a buffer exchangeprocess. In some embodiments, at least a portion of the receptortargeting conjugate is combined with the one or more lipids concurrentlywith the mixing step. In some embodiments, at least a portion of thereceptor targeting conjugate is combined with the one or more lipidsprior to the mixing step. In some embodiments, the receptor targetingconjugate is combined with the one or more lipids in the secondsolution. In some embodiments, a portion of the receptor targetingconjugate is combined with the one or more lipids in the second solutionand a portion of the receptor targeting conjugate is combined with theone or more lipids after the mixing. In some embodiments, a portion ofthe receptor targeting conjugate is combined with the one or more lipidsin the second solution and a portion of the receptor targeting conjugateis combined with the one or more lipids after the incubating step. Insome embodiments, a portion of the receptor targeting conjugate iscombined with the one or more lipids in the second solution and aportion of the receptor targeting conjugate is combined with the one ormore lipids after the buffer exchange step. In some embodiments, themethod further comprises diluting the mixture produced by mixing thefirst and the second solutions by adding a dilution buffer. In someembodiments, the mixture is diluted inline. In some embodiments, thedilution buffer comprises at least a portion of the receptor targetingconjugate. In some embodiments, the dilution buffer comprises at least aportion of the stealth lipid. In some embodiments, the first solutioncomprises an aqueous buffer. In some embodiments, the second solutioncomprises ethanol. In some embodiments, the mixing comprises laminarmixing, vortex mixing, turbulent mixing, or a combination thereof. Insome embodiments, the mixing comprises cross-mixing. In someembodiments, the mixing comprises inline mixing. In some embodiments,the mixing comprises introducing at least a portion of the firstsolution through a first inlet channel and at least a portion of thesecond solution through a second inlet channel, and wherein an anglebetween the first inlet channel and the second inlet channel is fromabout 15 to 180 degrees. In some embodiments, the mixing comprisesintroducing a portion of the first solution through a third inletchannel. In some embodiments, the buffer exchange comprises dialysis,chromatography, or tangential flow filtration (TFF). In someembodiments, the method further comprises a filtration step. In someembodiments, the receptor targeting conjugate comprises one or moreN-acetylgalactosamine (GalNAc) or GalNAc derivatives. In someembodiments, the receptor targeting conjugate comprises one or moregalactose or galactose derivatives. In some embodiments, the receptortargeting conjugate is selected from Table 4. In some embodiments, thereceptor targeting conjugate is a targeting conjugate described herein.In some embodiments, the nanoparticles comprise a first nanoparticlecomposition described herein. In some embodiments, the formulation is apharmaceutical composition described herein.

In one aspect, described herein is a pharmaceutical compositioncomprising nanoparticles, wherein the nanoparticles comprise (i) one ormore nucleic acid molecular entities, (ii) one or more lipids selectedfrom a sterol or a derivative thereof, a phospholipid, a stealth lipid,and an amino lipid, and (iii) a receptor targeting conjugate, whereinthe formulation is prepared by a method described herein. In one aspect,described herein is a pharmaceutical composition comprisingnanoparticles, wherein the nanoparticles comprise (i) one or morenucleic acid molecular entities, (ii) one or more lipids selected from asterol or a derivative thereof, a phospholipid, a stealth lipid, and anamino lipid, and (iii) a receptor targeting conjugate, wherein theformulation is prepared by a method comprising: (a) providing a firstsolution comprising the one or more nucleic acid molecular entities; (b)providing a second solution comprising at least one of the one or morelipids; (c) mixing the first solution and the second solution, therebyproducing a mixture comprising nanoparticles that comprise the one ormore nucleic acid molecular entities and the one or more lipids; (d)mixing the receptor targeting conjugate with the nanoparticles producedin step (c); (e) incubating the nanoparticles; and (f) optionallycarrying out a buffer exchange process. In one aspect, described hereinis a pharmaceutical composition comprising nanoparticles, wherein thenanoparticles comprise (i) one or more nucleic acid molecular entities,(ii) one or more lipids selected from a sterol or a derivative thereof,a phospholipid, a stealth lipid, and an amino lipid, and (iii) areceptor targeting conjugate, wherein the formulation is prepared by amethod comprising: (a) providing a first solution comprising the one ormore nucleic acid molecular entities; (b) providing a second solutioncomprising at least one of the one or more lipids; (c) mixing the firstsolution and the second solution, thereby producing a mixture comprisingnanoparticles that comprise the one or more nucleic acid molecularentities and the one or more lipids; (d) combining the receptortargeting conjugate with the one or more lipids, wherein at least aportion of the receptor targeting conjugate is combined with the one ormore lipids prior to or concurrently with the mixing step; (e)optionally incubating the nanoparticles; and (f) optionally carrying outa buffer exchange process. In one aspect, described herein is apharmaceutical composition comprising nanoparticles, wherein thenanoparticles comprise (i) one or more nucleic acid molecular entities,(ii) one or more lipids selected from a sterol or a derivative thereof,a phospholipid, a stealth lipid, and an amino lipid, and (iii) areceptor targeting conjugate, wherein the formulation is prepared by amethod comprising: (a) providing a first solution comprising the one ormore nucleic acid molecular entities; (b) providing a second solutioncomprising at least one of the one or more lipids; (c) inline mixing ofthe first solution and the second solution, thereby producing a mixturecomprising nanoparticles that comprise the one or more nucleic acidmolecular entities and the one or more lipids; (d) inline mixing of thereceptor targeting conjugate to the mixture of step (c), therebyproducing a mixture comprising nanoparticles that comprise the one ormore nucleic acid molecular entities, the one or more lipids, and thereceptor targeting conjugate; (e) diluting the mixture of step (d) byadding a dilution buffer; and (f) optionally carrying out a bufferexchange process. In one aspect, described herein is a pharmaceuticalcomposition comprising nanoparticles, wherein the nanoparticles comprise(i) one or more nucleic acid molecular entities, (ii) one or more lipidsselected from a sterol or a derivative thereof, a phospholipid, astealth lipid, and an amino lipid, and (iii) a receptor targetingconjugate, wherein the formulation is prepared by a method comprising:(a) providing a first solution comprising the one or more nucleic acidmolecular entities; (b) providing a second solution comprising (i) atleast one of the one or more lipids and (ii) at least a portion of thereceptor targeting conjugate; (c) mixing the first solution and thesecond solution, thereby producing a mixture comprising nanoparticles;(d) optionally incubating the nanoparticles; and optionally carrying outa buffer exchange process.

INCORPORATION BY REFERENCE

All publications, references, patents, and patent applications mentionedin this specification are herein incorporated by reference to the sameextent as if each individual publication, patent, or patent applicationwas specifically and individually indicated to be incorporated byreference. In the event of inconsistent usages between this document andthose documents so incorporated by reference, the usage in theincorporated reference(s) should be considered supplementary to that ofthis document; for irreconcilable inconsistencies, the usage in thisdocument controls

BRIEF DESCRIPTION OF THE DRAWINGS

Novel features of the invention are set forth with particularity in theappended claims. A better understanding of the features and advantagesof the present invention will be obtained by reference to the followingdetailed description that sets forth illustrative embodiments in whichthe principles of the inventions are utilized, and the accompanydrawings of which:

FIG. 1A-FIG. 1B illustrate the HPLC chromatogram of GalNAc-lipidincorporation of compositions herein. FIG. 1A shows reference LNP withno GalNAc-lipid present and FIG. 1B shows LNP constituted withGalNAc-lipid.

FIG. 2 illustrates in vitro PCSK9 gene editing efficiency in primaryhuman hepatocytes of LNP formulations in compositions herein.

FIG. 3 illustrates PCSK9 gene editing in wild type, LDLr−/−, and ApoE−/−mice liver, after retro-orbital administration of LNPs compositions herewithin, carrying SpCas9 mRNA and PCSK9 gRNA at 1:1 ratio.

FIG. 4 illustrates ANGPTL3 gene editing in LDLr−/− mice liver afterretro-orbital administration of LNPs compositions herein carrying ABEmRNA and ANGPTL3 gRNA at 1:1 ratio.

FIG. 5 illustrates PCSK9 gene editing in wild type and LDLr−/− miceliver after retro-orbital administration of LNPs carrying ABE mRNA andPCSK9 gRNA at 1:1 ratio.

FIG. 6 illustrates PCSK9 gene editing in wild type female micehepatocytes after retro-orbital administration of LNPs compositionsherein.

FIG. 7 illustrates PCSK9 gene editing in wild type female micehepatocytes after retro-orbital administration of LNPs compositionsherein.

FIG. 8 illustrates PCSK9 editing in LDLR−/− female mice hepatocytesafter retro-orbital administration of LNPs compositions herein carryingCas9 mRNA and gRNA.

FIG. 9 illustrates four general processes of introducing GalNAc-lipidsinto lipid nanoparticles.

FIG. 10 illustrates three protocols for preparing lipid nanoparticlescomprising post-addition of GalNAc-lipids.

FIG. 11 illustrates three protocols for preparing lipid nanoparticlescomprising post-addition of GalNAc-lipids.

FIG. 12 illustrates three protocols for preparing lipid nanoparticlescomprising addition of GalNAc-lipid into LNP excipients and splitaddition of GalNAc-Lipid.

FIG. 13 illustrates two protocols for preparing lipid nanoparticlescomprising addition of GalNAc-lipid into LNP excipients and splitaddition of GalNAc-Lipid.

FIG. 14 illustrates two protocols for preparing lipid nanoparticlescomprising cross-mixing of GalNAc-lipid.

FIG. 15 illustrates PCSK9 editing in LDLR−/− female mice hepatocytesafter retro-orbital administration of LNP compositions herein carryingPCSK9 ABE mRNA and guide RNA in a 1:1 ratio.

DETAILED DESCRIPTION

Certain specific details of this description are set forth in order toprovide a thorough understanding of various embodiments. However, oneskilled in the art will understand that the present disclosure may bepracticed without these details. In other instances, well-knownstructures and/or methods have not been shown or described in detail toavoid unnecessarily obscuring descriptions of the embodiments. Unlessthe context requires otherwise, throughout the specification and claimswhich follow, the word “comprise” and variations thereof, such as,“comprises” and “comprising” are to be construed in an open, inclusivesense, that is, as “including, but not limited to.” Further, headingsprovided herein are for convenience only and do not interpret the scopeor meaning of the claimed disclosure. The section headings used hereinare for organizational purposes only and are not to be construed aslimiting the subject matter described.

Efficient delivery to cells requires specific targeting and substantialprotection from the extracellular environment, particularly serumproteins. One method of achieving specific targeting is to conjugate atargeting moiety to active agents or pharmaceutical effector such as anucleic acid agent, thereby directing the active agent or pharmaceuticaleffector to particular cells or tissues depending on the specificity ofthe targeting moiety. One way a targeting moiety can improve delivery isby receptor mediated endocytotic activity. In some cases, this mechanismof uptake can involve the movement of nucleic acid agent bound tomembrane receptors into the interior of an area that is enveloped by themembrane via invagination of the membrane structure or by fusion of thedelivery system with the cell membrane. This process is initiated viaactivation of a cell-surface or membrane receptor following binding of aspecific ligand to the receptor. Many receptor-mediated endocytoticsystems are known and have been studied, including those that recognizesugars such as galactose, mannose, mannose-6-phosphate, peptides andproteins such as transferrin, asialoglycoprotein, vitamin B12, insulinand epidermal growth factor (EGF). Lipophilic moieties, such ascholesterol or fatty acids, when attached to highly hydrophilicmolecules such as nucleic acids can substantially enhance plasma proteinbinding and consequently circulation half life. Lipophilic conjugatescan also be used in combination with the targeting ligands in order toimprove the intracellular trafficking of a targeted delivery approach.

The Asialoglycoprotein receptor (ASGP-R) is a high capacity receptor,which is highly abundant on hepatocytes. The ASGP-R shows a 50-foldhigher affinity for N-Acetyl-D-Galactosylamine (GalNAc) than D-Gal.Previous work has shown that multivalency is required to achieve highaffinity, while spacing among sugars is also crucial. The inventors hererecognized that there is a clear need for new receptor specific ligandconjugated RNA or DNA agents and methods for their preparation, thataddress the shortcomings of in vivo delivery of therapeutics withnucleic acids or nucleic acid involved complexes as described above. Thepresent disclosure is directed to this very important objective.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural referents unless the contentclearly dictates otherwise. It should also be noted that the term “or”is generally employed in its sense including “and/or” unless the contentclearly dictates otherwise.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present disclosure, suitable methods andmaterials are described below. All references cited herein areincorporated by reference in their entirety as though fully set forth.Singleton et al., Dictionary of Microbiology and Molecular Biology 3rded., J. Wiley & Sons (New York, N.Y. 2001); March, Advanced OrganicChemistry Reactions, Mechanisms and Structure 5th ed., J. Wiley & Sons(New York, N.Y. 2001); and Sambrook and Russel, Molecular Cloning: ALaboratory Manual 3rd ed., Cold Spring Harbor Laboratory Press (ColdSpring Harbor, N.Y. 2001), provide one skilled in the art with a generalguide to many of the terms used in the present application.

Specific Definitions

When indicating the number of substituents, the term “one or more”refers to the range from one substituent to the highest possible numberof substitution, e.g. replacement of one hydrogen up to replacement ofall hydrogens by substituents.

The term “optional” or “optionally” denotes that a subsequentlydescribed event or circumstance can but need not occur, and that thedescription includes instances where the event or circumstance occursand instances in which it does not.

The term “nucleic acid molecular entity” is used interchangeably with“nucleic acid.”

The term “nucleic acid” as used herein generally refers to one or morenucleobases, nucleosides, or nucleotides, and the term includespolynucleobases, polynucleosides, and polynucleotides. A nucleic acidcan include polynucleotides, mononucleotides, and oligonucleoitdes. Anucleic acid can include DNA, RNA, or a mixture thereof, and can besingle stranded, double stranded, or partially single or doublestranded, and can form secondary structures. In some embodiments, anucleic acid has multiple double-stranded segments and single strandedsegments. For example, a nucleic acid may comprise a polynucleotide,e.g. a mRNA, with multiple double stranded segments within it. DNA maybe in the form of, e.g., antisense molecules, plasmid DNA, pre-condensedDNA, a PCR product, vectors, expression cassettes, chimeric sequences,chromosomalDNA, or derivatives and combinations of these groups. RNA maybe in the form of siRNA, asymmetrical interfering RNA (aiRNA), microRNA(miRNA), mRNA, tRNA, rRNA, tRNA, viral RNA (vRNA), CRISPR RNA, baseeditor RNA and combinations thereof. Nucleic acids include nucleic acidscontaining known nucleotide analogs or modified backbone residues orlinkages, which are synthetic, naturally occurring, and non-naturallyoccurring, and which have similar binding properties as the referencenucleic acid. Examples of such analogs include, without limitation,phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methylphosphonates, 2′-O-methyl ribonucleotides, and peptide-nucleic acids(PNAs). Unless specifically limited, the term encompasses nucleic acidscontaining known analogues of natural nucleotides that have similarbinding properties as the reference nucleic acid. Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (e.g., degeneratecodon substitutions), alleles, orthologs, SNPs, and complementarysequences as well as the sequence explicitly indicated. Specifically,degenerate codon substitutions may be achieved by generating sequencesin which the third position of one or more selected (or all) codons issubstituted with mixed-base and/or deoxyinosine residues (Batzer et al.,Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J Biol. Chem.,260:2605-2608 (1985); Rossolini et al., Mal. Cell. Probes, 8:91-98(1994)). “Nucleotides” contain a substituted and/or unsubstituted sugardeoxyribose (DNA), or a substituted and/or unsubstituted sugar ribose(RNA), or a substituted and/or unsubstituted carbocylic, or asubstituted and/or unsubstituted acyclic moiety (glycol nucleic, fore,g.), a base, and a phosphate group. Nucleotides are linked togetherthrough the phosphate groups. “Bases” include purines and pyrimidines,which further include natural compounds adenine, thymine, guanine,cytosine, uracil, inosine, and natural analogs, and syntheticderivatives of purines and pyrimidines, which include, but are notlimited to, modifications which place new reactive groups such as, butnot limited to, amines, alcohols, thiols, carboxylates, andalkylhalides.

The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequencethat comprises partial length or entire length coding sequencesnecessary for the production of a polypeptide or precursor polypeptide.

“Gene product,” as used herein, refers to a product of a gene such as anRNA transcript or a polypeptide.

The term “polynucleotide”, as used herein generally refers to a moleculecomprising two or more linked nucleic acid subunits, e.g., nucleotides,and can be used interchangeably with “oligonucleotide”. For example, apolynucleotide may include one or more nucleotides selected fromadenosine (A), cytosine (C), guanine (G), thymine (T) and uracil (U), orvariants thereof. A nucleotide generally includes a nucleoside and atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphate (P03) groups. Anucleotide can include a nucleobase, a five-carbon sugar (either riboseor deoxyribose), and one or more phosphate groups. Ribonucleotidesinclude nucleotides in which the sugar is ribose. Deoxyribonucleotidesinclude nucleotides in which the sugar is deoxyribose. A nucleotide canbe a nucleoside monophosphate, nucleoside diphosphate, nucleosidetriphosphate or a nucleoside polyphosphate. For example, a nucleotidecan be a deoxyribonucleoside polyphosphate, such as adeoxyribonucleoside triphosphate (dNTP), Exemplary dNTPs includedeoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP),deoxyguanosine triphosphate (dGTP), uridine triphosphate (dUTP) anddeoxythymidine triphosphate (dTTP). dNTPs can also include detectabletags, such as luminescent tags or markers (e.g., fluorophores). Forexample, a nucleotide can be a purine (e.g., A or G, or variant thereof)or a pyrimidine (e.g., C, T or U, or variant thereof). In some examples,a polynucleotide is deoxyribonucleic acid (DNA), ribonucleic acid (RNA),or derivatives or variants thereof. Exemplary polynucleotides include,but are not limited to, short interfering RNA (siRNA), a microRNA(miRNA), a plasmid DNA (pDNA), a short hairpin RNA (shRNA), smallnuclear RNA (snRNA), messenger RNA (mRNA), precursor mRNA (pre-mRNA),antisense RNA (asRNA), and heteronuclear RNA (hnRNA), and encompassesboth the nucleotide sequence and any structural embodiments thereof,such as single-stranded, double-stranded, triple-stranded, helical,hairpin, stem loop, bulge, etc. In some cases, a polynucleotide iscircular. A polynucleotide can have various lengths. For example, apolynucleotide can have a length of at least about 7 bases, 8 bases, 9bases, 10 bases, 20 bases, 30 bases, 40 bases, 50 bases, 100 bases, 200bases, 300 bases, 400 bases, 500 bases, 1 kilobase (kb), 2 kb, 3, kb, 4kb, 5 kb, 10 kb, 50 kb, or more. A polynucleotide can be isolated from acell or a tissue. For example, polynucleotide sequences may compriseisolated and purified DNA/RNA molecules, synthetic DNA/RNA molecules,and/or synthetic DNA/RNA analogs.

Polynucleotides can include one or more nucleotide variants, includingnonstandard nucleotide(s), non-natural nucleotide(s), nucleotideanalog(s) and/or modified nucleotides. Examples of modified nucleotidesinclude, but are not limited to diaminopurine, 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine,4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine,N6-isopentenyladenine, 1-methylguanine, 1-methylinosine,2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine,5-methylcytosine, N6-adenine, 7-methylguanine,5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, 5-methyl-2-thiouracil,3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, 2,6-diaminopurine andthe like. In some cases, nucleotides may include modifications in theirphosphate moieties, including modifications to a triphosphate moiety.Non-limiting examples of such modifications include phosphate chains ofgreater length (e.g., a phosphate chain having, 4, 5, 6, 7, 8, 9, 10 ormore phosphate moieties) and modifications with thiol moieties (e.g.,alpha-thiotriphosphate and beta-thiotriphosphates). Nucleic acidmolecules may also be modified at the base moiety (e.g., at one or moreatoms that typically are available to form a hydrogen bond with acomplementary nucleotide and/or at one or more atoms that are nottypically capable of forming a hydrogen bond with a complementarynucleotide), sugar moiety or phosphate backbone. Nucleic acid moleculesmay also contain amine-modified groups, such as amino ally 1-dUTP(aa-dUTP) and aminohexhylacrylamide-dCTP (aha-dCTP) to allow covalentattachment of amine reactive moieties, such as N-hydroxysuccinimideesters (NHS). Alternatives to standard DNA base pairs or RNA base pairsin the oligonucleotides of the present disclosure can provide higherdensity in bits per cubic mm, higher safety (resistant to accidental orpurposeful synthesis of natural toxins), easier discrimination inphoto-programmed polymerases, or lower secondary structure. Suchalternative base pairs compatible with natural and mutant polymerasesfor de novo and/or amplification synthesis are described in Betz K,Malyshev D A, Lavergne T, Welte W, Diederichs K, Dwyer T J, OrdoukhanianP, Romesberg F E, Marx A. Nat. Chem. Biol. 2012 July; 8(7):612-4, whichis herein incorporated by reference for all purposes.

As used herein, the terms “polypeptide”, “protein” and “peptide” areused interchangeably and refer to a polymer of amino acid residueslinked via peptide bonds and which may be composed of two or morepolypeptide chains. The terms “polypeptide”, “protein” and “peptide”refer to a polymer of at least two amino acid monomers joined togetherthrough amide bonds. An amino acid may be the L-optical isomer or theD-optical isomer. More specifically, the terms “polypeptide”, “protein”and “peptide” refer to a molecule composed of two or more amino acids ina specific order; for example, the order as determined by the basesequence of nucleotides in the gene or RNA coding for the protein.Proteins are essential for the structure, function, and regulation ofthe body's cells, tissues, and organs, and each protein has uniquefunctions. Examples are hormones, enzymes, antibodies, and any fragmentsthereof. In some cases, a protein can be a portion of the protein, forexample, a domain, a subdomain, or a motif of the protein. In somecases, a protein can be a variant (or mutation) of the protein, whereinone or more amino acid residues are inserted into, deleted from, and/orsubstituted into the naturally occurring (or at least a known) aminoacid sequence of the protein. A protein or a variant thereof can benaturally occurring or recombinant.

As used herein, the term “intercalating” or “intercalation” refers tothe actions of agents (e.g., small molecules) that insert themselvesbetween successive bases in DNA. In some cases, the intercalationprevents the proper functioning of the DNA.

As used herein, “complement” means the complementary sequence to anucleic acid according to standard Watson/Crick pairing rules. Acomplement sequence can also be a sequence of RNA complementary to theDNA sequence or its complement sequence, and can also be a cDNA.Complements may be fully complementary or partially complementary suchthat the two sequences will hybridize under stringent hybridizationconditions. The skilled artisan will understand that complementary orsubstantially complementary sequences need not hybridize along theirentire length. In particular embodiments, complementary or substantiallycomplementary sequences may comprise a contiguous sequence of bases thatdo not hybridize to a target sequence, positioned 3′ or 5′ to acontiguous sequence of bases that hybridize to a target sequence.

As used herein, “hybridize” refers to a process where two nucleic acidstrands anneal to each in accordance with Watson-Crick base pairingrules. Nucleic acid hybridization techniques are well known in the art.See, e.g., Sambrook, et al., 1989, Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Press, Plainview, N.Y. Thoseskilled in the art understand how to determine the appropriatestringency of hybridization/washing conditions such that sequenceshaving at least a desired level of complementarity will stablyhybridize, while those having lower complementarity will not. Forexamples of hybridization conditions and parameters, see, e.g.,Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Press, Plainview, N.Y.; Ausubel, F. M. etal. 1994, Current Protocols in Molecular Biology. John Wiley & Sons,Secaucus, N.J., all of which are incorporated herein by reference intheir entireties. In certain embodiments, hybridizations may occurbetween nucleic acid molecules of 20-100 nucleotides in length. In someembodiments, hybridization may occur between at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, or 100 consecutive nucleotides. In some embodiments, thehybridizing nucleic acid molecules may contain up to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mismatches thatare tolerated.

As used herein, the term “biological sample” means any biologicalmaterial from which polynucleotides, polypeptides, biomarkers, and/ormetabolites can be prepared and examined. Non-limiting examplesencompasses whole blood, plasma, saliva, cheek swab, fecal specimen,urine specimen, cell mass, or any other bodily fluid or tissue.

The terms “administer,” “administering”, “administration,” and the like,as used herein, refer to the methods that may be used to enable deliveryof compounds or compositions to the desired site of biological action.These methods include, but are not limited to oral routes (p.o.),intraduodenal routes (i.d.), parenteral injection (including intravenous(i.v.), subcutaneous (s.c.), intraperitoneal (i.p.), intramuscular(i.m.), intravascular or infusion (inf.)), topical (top.) and rectal(p.r.) administration. Those of skill in the art are familiar withadministration techniques that can be employed with the compounds andmethods described herein. In some embodiments, the compounds andcompositions described herein are administered orally.

The terms “co-administration” or the like, as used herein, are meant toencompass administration of the selected therapeutic agents to a singlepatient, and are intended to include treatment regimens in which theagents are administered by the same or different route of administrationor at the same or different time.

The terms “effective amount” or “therapeutically effective amount,” asused herein, refer to a sufficient amount of an agent or a compoundbeing administered which will relieve to some extent one or more of thesymptoms of the disease or condition being treated; for example areduction and/or alleviation of one or more signs, symptoms, or causesof a disease, or any other desired alteration of a biological system.For example, an “effective amount” for therapeutic uses can be an amountof an agent that provides a clinically significant decrease in one ormore disease symptoms. An appropriate “effective” amount may bedetermined using techniques, such as a dose escalation study, inindividual cases.

The terms “enhance” or “enhancing,” as used herein, means to increase orprolong either in amount, potency or duration a desired effect.

As used herein, “carbohydrate” refers to a compound which is either acarbohydrate per se made up of one or more monosaccharide units havingat least 6 carbon atoms (which may be linear, branched or cyclic) withan oxygen, nitrogen or sulfur atom bonded to each carbon atom; or acompound having as a part thereof a carbohydrate moiety made up of oneor more monosaccharide units each having at least six carbon atoms(which may be linear, branched or cyclic), with an oxygen, nitrogen orsulfur atom bonded to each carbon atom. Representative carbohydratesinclude the sugars (mono-, di-, tri- and oligosaccharides containingfrom about 4-9 monosaccharide units), and polysaccharides such asstarches, glycogen, cellulose and polysaccharide gums. Specificmonosaccharides include C5 and above (preferably C5-C8) sugars; di- andtrisaccharides include sugars having two or three monosaccharide units(preferably C5-C8).

The term “monosaccharide” embraces radicals of allose, altrose,arabinose, cladinose, erythrose, erythrulose, fructose, D-fucitol,L-fucitol, fucosamine, fucose, fuculose, galactosamine,D-galactosaminitol, N-acetyl-galctosamine, galactose, glucosamine,N-acetyl-glucosamine, glucosaminitol, glucose, glucose-6-phosphategulose glyceraldehyde, L-glycero-D-mannos-heprose, glycerol, glycerone,gulose idose, lyxose, mannosamine, mannose, mannose-6-phosphate,psicose, quinovose, quinovosamine, rhamnitol, rhamnosamine, rhamnose,ribose, ribulose, sedoheptulose, sorbose, tagatose, talose, tartaricacid, throse, xylose and xylulose. The monosaccharide can be in D- orL-configuration. The monosaccharide may further be a deoxy sugar(alcoholic hydroxy group replaced by hydrogen), amino sugar (alcoholichydroxy group replaced by amino group), a thio sugar (alcoholic hydroxygroup replaced by thiol, or C═O replaced by C═S, or a ring oxygen ofcyclic form replaced by sulfur), a seleno sugar, a telluro sugar, an azasugar (ring carbon replaced by nitrogen), a imino sugar (ring oxygenreplaced by nitrogen), a phosphano sugar (ring oxygen replaced withphosphorus), a phospha sugar (ring carbon replaced with phosphorus), aC-substituted monosaccharide (hydrogen at a non-terminal carbon atomreplaced with carbon), an unsaturated monosaccharide, an alditol(carbonyl group replaced with CHOH group), aldonic acid (aldehydic groupreplaced by carboxy group), a ketoaldonic acid, a uronic acid, analdaric acid, and so forth. Amino sugars include amino monosaccharides,preferably galactosamine, glusamine, mannosamine, fucosmine,quinavosamine, neuraminic acid, muramic acid, lactosediamine, acosamine,bacillosamine, daunosamine, desosamine, forosamine, garosamine,kanosamine, kanosamine, mycaminose, myosamine, persosamine,pneumosamine, purpurosamine, rhodosmine. It is understood that themonosaccharide and the like can be further substituted.

As used herein, the “N/P ratio” is the molar ratio of ionizable (e.g.,in the physiological pH range) nitrogen atoms in a lipid (or lipids) tophosphate groups in a nucleic acid molecular entity (or nucleic acidmolecular entities), e.g., in a nanoparticle composition comprising alipid component and an RNA. Ionizable nitrogen atoms can include, forexample, nitrogen atoms that can be protonated at about pH 1, about pH2, about pH 3, about pH 4, about pH5, about pH 6, about pH 7, about pH7.5, or about pH 8 or higher. The physiological pH range can include,for example, the pH range of different cellular compartments (such asorgans, tissues, and cells) and bodily fluids (such as blood, CSF,gastric juice, milk, bile, saliva, tears, and urine). In certainspecific embodiments, the physiological pH range refers to the pH rangeof blood in a mammal, for example, from about 7.35 to about 7.45. Insome embodiments, ionizable nitrogen atoms refer to those nitrogen atomsthat are ionizable within a pH range between 5 and 14.

The terms “disaccharide”, “trisaccharide” and “polysaecharide” embraceradicals of abequose, acrabose, amicetose, amylopectin, amylose, apiose,arcanose, ascarylose, ascorbic acid, boivinose, cellobiose, cellotriose,cellulose, chacotriose, chalcose, chitin, colitose, cyclodextrin,cymarose, dextrin, 2-deoxyribose, 2-deoxyglucose diginose, digitalose,digitoxose, evalose, evemitrose, fructooligosachharide,galto-oligosaccharide, gentianose, genitiobiose, glucan, gluicogen,glylcogen, hamamelose, heparin, inulin, isolevoglucosenone, isomaltose,isomaltotriose, isopanose, kojibiose, lactose, lactosamine,lactosediamine, laminarabiose, levoglucosan, levoglucosenone, β-maltose,maltriose, mannan-oligosacchardie, amnninotriose, melezitose, melibiose,muramic acid, mycarose, mycinose, neuaminic acid, migerose, nojirimycon,noviose, oleandrose, panose, paratose, planteose, primeverose,raffinose, rhodone, rutinose, oleandrose, panose, paratose, planteose,primeverose, raffinose, rhodinose, rutinose, sarmentose, sedoheptulose,sedoheptulosan, solatriose, sophorose, stachyose, streptose, sucrose,α,α-trehalose, trahalosamine, turanose, tyvelose, xylobiose,umbelliferose and the like. Further, it is understood that the“disaccharide”, “trisaccharide” and “polysaccharide” and the like canfurther substituted. Disaccharide also includes amino sugars and theirderivatives, particularly, a mycaminose derivatized a the C-4′ positionor a 4 deoxy-3-amino-glucose derivatized at the C-6′ position.

The term “subject” or “patient” encompasses mammals. Examples of mammalsinclude, but are not limited to, any member of the mammalian class:humans, non-human primates such as chimpanzees, and other apes andmonkey species; farm animals such as cattle, horses, sheep, goats,swine; domestic animals such as rabbits, dogs, and cats; laboratoryanimals including rodents, such as rats, mice and guinea pigs, and thelike. In one aspect, the mammal is a human. The term “animal” as usedherein comprises human beings and non-human animals. In one embodiment,a “non-human animal” is a mammal, for example a rodent such as rat or amouse. In one embodiment, a non-human animal is a mouse or a monkey.

The terms “treat,” “treating” or “treatment,” as used herein, includealleviating, abating or ameliorating at least one symptom of a diseaseor condition, preventing additional symptoms, inhibiting the disease orcondition, e.g., arresting the development of the disease or condition,relieving the disease or condition, causing regression of the disease orcondition, relieving a condition caused by the disease or condition, orstopping the symptoms of the disease or condition eitherprophylactically and/or therapeutically. The term “treating” furtherencompasses the concept of “prevent,” “preventing,” and “prevention,” asstated below. It is appreciated that, although not precluded, treating adisorder or condition does not require that the disorder, condition, orsymptoms associated therewith be completely eliminated.

The term “preventing” or “prevention” of a disease state denotes causingthe clinical symptoms of the disease state not to develop in a subjectthat can be exposed to or predisposed to the disease state, but does notyet experience or display symptoms of the disease state.

The terms “pharmaceutical composition” and “pharmaceutical formulation”(or “formulation”) are used interchangeably and denote a mixture orsolution comprising a therapeutically effective amount of an activepharmaceutical ingredient together with one or more pharmaceuticallyacceptable excipients to be administered to a subject, e.g., a human inneed thereof.

The term “pharmaceutical combination” as used herein, means a productthat results from mixing or combining more than one active ingredientand includes both fixed and non-fixed combinations of the activeingredients. The term “fixed combination” means that the activeingredients, e.g., a compound described herein and a co-agent, are bothadministered to a patient simultaneously in the form of a single entityor dosage. The term “non-fixed combination” means that the activeingredients, e.g. a compound described herein and a co-agent, areadministered to a patient as separate entities either simultaneously,concurrently or sequentially with no specific intervening time limits,wherein such administration provides effective levels of the twocompounds in the body of the patient. The latter also applies tococktail therapy, e.g., administration of three or more activeingredients.

The term “pharmaceutically acceptable” denotes an attribute of amaterial which is useful in preparing a pharmaceutical composition thatis generally safe, non-toxic, and neither biologically nor otherwiseundesirable and is acceptable for veterinary as well as humanpharmaceutical use. “Pharmaceutically acceptable” can refer to amaterial, such as a carrier or diluent, which does not abrogate thebiological activity or properties of the compound, and is relativelynontoxic, e.g., the material may be administered to an individualwithout causing undesirable biological effects or interacting in adeleterious manner with any of the components of the composition inwhich it is contained.

The terms “pharmaceutically acceptable excipient”, “pharmaceuticallyacceptable carrier”, “pharmaceutically acceptable vehicle” and“therapeutically inert excipient” can be used interchangeably and denoteany pharmaceutically acceptable ingredient in a pharmaceuticalcomposition having no therapeutic activity and being non-toxic to thesubject administered, such as disintegrators, binders, fillers,solvents, buffers, tonicity agents, stabilizers, antioxidants,surfactants, carriers, diluents, excipients, preservatives or lubricantsused in formulating pharmaceutical products.

The term “base editing” and “base correction” are used interchangeablyto indicate a base change or mutation at a target sequence within thetarget gene leading to base modification. In certain embodiments, baseediting occurs at a single base of the target sequence. In preferredembodiments, base editing does not involve double strand breaks of thetarget sequence.

As used herein, the term “siRNA” refers to an agent that mediates thetargeted cleavage of an RNA transcript. These agents associate with acytoplasmic multi-protein complex known as RNAi-induced silencingcomplex (RISC). Agents that are effective in inducing RNA interferenceare also referred to as siRNA, RNAi agent, or iRNA agent, herein. Asused herein, the term siRNA includes microRNAs and pre-microRNAs. Asused herein, the terms “siRNA activity” and “RNAi activity” refer togene silencing by an siRNA.

The term “2′-O-methoxyethyl” (also 2′-MOE, 2′-O(CH₂)₂—OCH₃ and2′-O-(2-methoxyethyl)) refers to an O-methoxy-ethyl modification of the2′ position of a furosyl ring. A 2′-O-methoxyethyl modified sugar is amodified sugar.

The term “2′-O-methoxyethyl nucleotide” means a nucleotide comprising a2′-O-methoxyethyl modified sugar moiety.

The term “5-methylcytosine” means a cytosine modified with a methylgroup attached to the 5′ position. A 5-methylcytosine is a modifiednucleobase.

The term “oxo” refers to the =0 substituent.

The term “alkyl” refers to a straight or branched hydrocarbon chainradical, having from one to twenty carbon atoms, and which is attachedto the rest of the molecule by a single bond. An alkyl comprising up to10 carbon atoms is referred to as a C₁-C₁₀ alkyl, likewise, for example,an alkyl comprising up to 6 carbon atoms is a C₁-C₆ alkyl. Alkyls (andother moieties defined herein) comprising other numbers of carbon atomsare represented similarly. Alkyl groups include, but are not limited to,C₁-C₁₀ alkyl, C₁-C₉ alkyl, C₁-C₈ alkyl, C₁-C₇ alkyl, C₁-C₆ alkyl, C₁-C₅alkyl, C₁-C₄ alkyl, C₁-C₃ alkyl, C₁-C₂ alkyl, C₂-C₈ alkyl, C₃-C₈alkyland C₄-C₈ alkyl. Representative alkyl groups include, but are notlimited to, methyl, ethyl, n-propyl, 1-methylethyl (i-propyl), n-butyl,i-butyl, s-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl,2-methylhexyl, 1-ethyl-propyl, and the like. In some embodiments, thealkyl is methyl or ethyl. In some embodiments, the alkyl is —CH(CH₃)₂ or—C(CH₃)₃. Unless stated otherwise specifically in the specification, analkyl group may be optionally substituted as described below. “Alkylene”or “alkylene chain” refers to a straight or branched divalenthydrocarbon chain linking the rest of the molecule to a radical group.In some embodiments, the alkylene is —CH₂—, —CH₂CH₂—, or —CH₂CH₂CH₂—. Insome embodiments, the alkylene is —CH₂—. In some embodiments, thealkylene is —CH₂CH₂—. In some embodiments, the alkylene is —CH₂CH₂CH₂—.

The term “alkoxy” refers to a radical of the formula —OR where R is analkyl radical as defined. Unless stated otherwise specifically in thespecification, an alkoxy group may be optionally substituted asdescribed below. Representative alkoxy groups include, but are notlimited to, methoxy, ethoxy, propoxy, butoxy, pentoxy. In someembodiments, the alkoxy is methoxy. In some embodiments, the alkoxy isethoxy.

The term “alkylamino” refers to a radical of the formula —NHR or —NRRwhere each R is, independently, an alkyl radical as defined above.Unless stated otherwise specifically in the specification, an alkylaminogroup may be optionally substituted as described below.

The term “alkenyl” refers to a type of alkyl group in which at least onecarbon-carbon double bond is present. In one embodiment, an alkenylgroup has the formula —C(R)═CR₂, wherein R refers to the remainingportions of the alkenyl group, which may be the same or different. Insome embodiments, R is H or an alkyl. In some embodiments, an alkenyl isselected from ethenyl (i.e., vinyl), propenyl (i.e., allyl), butenyl,pentenyl, pentadienyl, and the like. Non-limiting examples of an alkenylgroup include —CH═CH₂, —C(CH₃)═CH₂, —CH═CHCH₃, —C(CH₃)═CHCH₃, and—CH₂CH═CH₂. Depending on the structure, an alkenyl group can bemonovalent or divalent (i.e., an alkenylene group).

The term “alkynyl” refers to a type of alkyl group in which at least onecarbon-carbon triple bond is present. Accordingly, “alkynylene” canrefer to a divalent alkynyl group. In one embodiment, an alkenyl grouphas the formula —C≡C—R, wherein R refers to the remaining portions ofthe alkynyl group. In some embodiments, R is H or an alkyl. In someembodiments, an alkynyl is selected from ethynyl, propynyl, butynyl,pentynyl, hexynyl, and the like. Non-limiting examples of an alkynylgroup include —C≡CH, —C≡CCH₃—C≡CCH₂CH₃, —CH₂C≡CH.

The term “aryl” refers to an aromatic ring wherein each of the atomsforming the ring is a carbon atom. Aryl groups can be optionallysubstituted. Examples of aryl groups include, but are not limited tophenyl, and naphthyl. In some embodiments, the aryl is phenyl. Dependingon the structure, an aryl group can be monovalent or divalent (i.e., an“arylene” group). Unless stated otherwise specifically in thespecification, the term “aryl” or the prefix “ar-” (such as in“aralkyl”) is meant to include aryl radicals that are optionallysubstituted. In some embodiments, an aryl group is partially reduced toform a cycloalkyl group defined herein. In some embodiments, an arylgroup is fully reduced to form a cycloalkyl group defined herein. Insome embodiments, an aryl group is a C₆-C₁₄ aryl. In some embodiments,an aryl group is a C₆-C₁₀ aryl.

The term “cycloalkyl” refers to a monocyclic or polycyclic non-aromaticradical, wherein each of the atoms forming the ring (i.e. skeletalatoms) is a carbon atom. In some embodiments, cycloalkyls are saturatedor partially unsaturated. In some embodiments, cycloalkyls arespirocyclic or bridged compounds. In some embodiments, cycloalkyls arefused with an aromatic ring (in which case the cycloalkyl is bondedthrough a non-aromatic ring carbon atom). Cycloalkyl groups includegroups having from 3 to 10 ring atoms. Representative cycloalkylsinclude, but are not limited to, cycloalkyls having from three to tencarbon atoms, from three to eight carbon atoms, from three to six carbonatoms, or from three to five carbon atoms. Monocyclic cycloalkylradicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, and cyclooctyl. In some embodiments, themonocyclic cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl orcyclohexyl. In some embodiments, the monocyclic cycloalkyl iscyclopentenyl or cyclohexenyl. In some embodiments, the monocycliccycloalkyl is cyclopentenyl. Polycyclic radicals include, for example,adamantyl, 1,2-dihydronaphthalenyl, 1,4-dihydronaphthalenyl, tetrainyl,decalinyl, 3,4-dihydronaphthalenyl-1(2H)-one, spiro[2.2]pentyl,norbornyl and bicycle[1.1.1]pentyl. Unless otherwise stated specificallyin the specification, a cycloalkyl group may be optionally substituted.Depending on the structure, a cycloalkyl group can be monovalent ordivalent (i.e., a cycloalkylene group).

The term “haloalkyl” denotes an alkyl group wherein at least one of thehydrogen atoms of the alkyl group has been replaced by same or differenthalogen atoms, particularly fluoro atoms. Examples of haloalkyl includemonofluoro-, difluoro- or trifluoro-methyl, -ethyl or -propyl, forexample 3,3,3-trifluoropropyl, 2-fluoroethyl, 2,2,2-trifluoroethyl,fluoromethyl, or trifluoromethyl. The term “perhaloalkyl” denotes analkyl group where all hydrogen atoms of the alkyl group have beenreplaced by the same or different halogen atoms.

The term “heteroalkylene” refers to an alkyl radical as described abovewhere one or more carbon atoms of the alkyl is replaced with a O, N or Satom. “Heteroalkylene” or “heteroalkylene chain” refers to a straight orbranched divalent heteroalkyl chain linking the rest of the molecule toa radical group. Unless stated otherwise specifically in thespecification, the heteroalkyl or heteroalkylene group may be optionallysubstituted as described below. Representative heteroalkylene groupsinclude, but are not limited to —OCH₂CH₂O—, —OCH₂CH₂OCH₂CH₂O—, or—OCH₂CH₂OCH₂CH₂OCH₂CH₂O—.

The term “heterocycloalkyl” refers to a cycloalkyl group that includesat least one heteroatom selected from nitrogen, oxygen, and sulfur.Unless stated otherwise specifically in the specification, theheterocycloalkyl radical may be a monocyclic, or bicyclic ring system,which may include fused (when fused with an aryl or a heteroaryl ring,the heterocycloalkyl is bonded through a non-aromatic ring atom) orbridged ring systems. The nitrogen, carbon or sulfur atoms in theheterocyclyl radical may be optionally oxidized. The nitrogen atom maybe optionally quaternized. The heterocycloalkyl radical is partially orfully saturated. Examples of heterocycloalkyl radicals include, but arenot limited to, dioxolanyl, thienyl[1,3]dithianyl, tetrahydroquinolyl,tetrahydroisoquinolyl, decahydroquinolyl, decahydroisoquinolyl,imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl,morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl,2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl,piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl,thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl,thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl,1,1-dioxo-thiomorpholinyl. The term heterocycloalkyl also includes allring forms of carbohydrates, including but not limited tomonosaccharides, disaccharides and oligosaccharides. Unless otherwisenoted, heterocycloalkyls have from 2 to 12 carbons in the ring. In someembodiments, heterocycloalkyls have from 2 to 10 carbons in the ring. Insome embodiments, heterocycloalkyls have from 2 to 10 carbons in thering and 1 or 2 N atoms. In some embodiments, heterocycloalkyls havefrom 2 to 10 carbons in the ring and 3 or 4 N atoms. In someembodiments, heterocycloalkyls have from 2 to 12 carbons, 0-2 N atoms,0-2 O atoms, 0-2 P atoms, and 0-1 S atoms in the ring. In someembodiments, heterocycloalkyls have from 2 to 12 carbons, 1-3 N atoms,0-1 O atoms, and 0-1 S atoms in the ring. It is understood that whenreferring to the number of carbon atoms in a heterocycloalkyl, thenumber of carbon atoms in the heterocycloalkyl is not the same as thetotal number of atoms (including the heteroatoms) that make up theheterocycloalkyl (i.e. skeletal atoms of the heterocycloalkyl ring).Unless stated otherwise specifically in the specification, aheterocycloalkyl group may be optionally substituted. As used herein,the term “heterocycloalkylene” can refer to a divalent heterocycloalkylgroup.

The term “heteroaryl” refers to an aryl group that includes one or morering heteroatoms selected from nitrogen, oxygen and sulfur. Theheteroaryl is monocyclic or bicyclic. Illustrative examples ofmonocyclic heteroaryls include pyridinyl, imidazolyl, pyrimidinyl,pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl,thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, pyridazinyl, triazinyl,oxadiazolyl, thiadiazolyl, furazanyl, indolizine, indole, benzofuran,benzothiophene, indazole, benzimidazole, purine, quinolizine, quinoline,isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline,1,8-naphthyridine, and pteridine. Illustrative examples of monocyclicheteroaryls include pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl,triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl,oxazolyl, isothiazolyl, pyrrolyl, pyridazinyl, triazinyl, oxadiazolyl,thiadiazolyl, and furazanyl. Illustrative examples of bicyclicheteroaryls include indolizine, indole, benzofuran, benzothiophene,indazole, benzimidazole, purine, quinolizine, quinoline, isoquinoline,cinnoline, phthalazine, quinazoline, quinoxaline, 1,8-naphthyridine, andpteridine. In some embodiments, heteroaryl is pyridinyl, pyrazinyl,pyrimidinyl, thiazolyl, thienyl, thiadiazolyl or furyl. In someembodiments, a heteroaryl contains 0-6 N atoms in the ring. In someembodiments, a heteroaryl contains 1-4 N atoms in the ring. In someembodiments, a heteroaryl contains 4-6 N atoms in the ring. In someembodiments, a heteroaryl contains 0-4 N atoms, 0-1 O atoms, 0-1 Patoms, and 0-1 S atoms in the ring. In some embodiments, a heteroarylcontains 1-4 N atoms, 0-1 O atoms, and 0-1 S atoms in the ring. In someembodiments, heteroaryl is a C₁-C₉ heteroaryl. In some embodiments,monocyclic heteroaryl is a C₁-C₅ heteroaryl. In some embodiments,monocyclic heteroaryl is a 5-membered or 6-membered heteroaryl. In someembodiments, a bicyclic heteroaryl is a C₆-C₉ heteroaryl. In someembodiments, a heteroaryl group is partially reduced to form aheterocycloalkyl group defined herein. In some embodiments, a heteroarylgroup is fully reduced to form a heterocycloalkyl group defined herein.Depending on the structure, a heteroaryl group can be monovalent ordivalent (i.e., a “heteroarylene” group).

The term “substituted,” “substituent” or the like, unless otherwiseindicated, can refer to the replacement of one or more hydrogen radicalsin a given structure individually and independently with the radical ofa specified substituent including, but not limited to: D, halogen, —CN,—NH₂, —NH(alkyl), —N(alkyl)₂, —OH, —CO₂H, —CO₂alkyl, —C(═O)NH₂,—C(═O)NH(alkyl), —C(═O)N(alkyl)₂, —S(═O)₂NH₂, —S(═O)₂NH(alkyl),—S(═O)₂N(alkyl)₂, alkyl, cycloalkyl, fluoroalkyl, heteroalkyl, alkoxy,fluoroalkoxy, heterocycloalkyl, aryl, heteroaryl, aryloxy, alkylthio,arylthio, alkylsulfoxide, arylsulfoxide, alkylsulfone, and arylsulfone.In some other embodiments, optional substituents are independentlyselected from D, halogen, —CN, —NH₂, —NH(CH₃), —N(CH₃)₂, —OH, —CO₂H,—CO₂(C₁-C₄ alkyl), —C(═O)NH₂, —C(═O)NH(C₁-C₄ alkyl), —C(═O)N(C₁-C₄alkyl)₂, —S(═O)₂NH₂, —S(═O)₂NH(C₁-C₄ alkyl), —S(═O)₂N(C₁-C₄ alkyl)₂,C₁-C₄ alkyl, C₃-C₆ cycloalkyl, C₁-C₄ fluoroalkyl, C₁-C₄ heteroalkyl,C₁-C₄ alkoxy, C₁-C₄ fluoroalkoxy, —SC₁—C₄ alkyl, —S(═O)C₁-C₄ alkyl, and—S(═O)₂(C₁-C₄ alkyl). In some embodiments, optional substituents areindependently selected from D, halogen, —CN, —NH₂, —OH, —NH(CH₃),—N(CH₃)₂, —NH(cyclopropyl), —CH₃, —CH₂CH₃, —CF₃, —OCH₃, and —OCF₃. Insome embodiments, substituted groups are substituted with one or two ofthe preceding groups. In some embodiments, an optional substituent on analiphatic carbon atom (acyclic or cyclic) includes oxo (═O).

The term “unsubstituted” means that the specified group bears nosubstituents. The term “optionally substituted” means that the specifiedgroup is unsubstituted or substituted by one or more substituents,independently chosen from the group of possible substituents. Whenindicating the number of substituents, the term “one or more” means fromone substituent to the highest possible number of substitution, i.e.replacement of one hydrogen up to replacement of all hydrogens bysubstituents.

“About” means within ±10% of a value. For example, if it is stated, “amarker may be increased by about 50%”, it is implied that the marker maybe increased between 45%-55%.

“Active pharmaceutical agent” means the substance or substances in apharmaceutical composition that provide a therapeutic benefit whenadministered to an individual.

“Dosage unit” means a form in which a pharmaceutical agent is provided,e.g. pill, tablet, or other dosage unit known in the art. In certainembodiments, a dosage unit is a vial containing lyophilized antisenseoligonucleotide. In certain embodiments, a dosage unit is a vialcontaining reconstituted antisense oligonucleotide.

“Dose” means a specified quantity of a pharmaceutical agent provided ina single administration, or in a specified time period. In certainembodiments, a dose can be administered in one, two, or more boluses,tablets, or injections. For example, in certain embodiments wheresubcutaneous administration is desired, the desired dose requires avolume not easily accommodated by a single injection, therefore, two ormore injections can be used to achieve the desired dose. In certainembodiments, the pharmaceutical agent is administered by infusion overan extended period of time or continuously. Doses can be stated as theamount of pharmaceutical agent per hour, day, week, or month. Doses canalso be stated as mg/kg or g/kg.

“Modified internucleoside linkage” refers to a substitution or anychange from a naturally occurring internucleoside bond. For example, aphosphorothioate linkage is a modified internucleoside linkage.

“Modified nucleobase” refers to any nucleobase other than adenine,cytosine, guanine, thymidine, or uracil. For example, 5-methylcytosineis a modified nucleobase. An “unmodified nucleobase” means the purinebases adenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C), and uracil (U).

“Modified nucleoside” means a nucleoside having at least one modifiedsugar moiety, and/or modified nucleobase.

“Modified nucleotide” means a nucleotide having at least one modifiedsugar moiety, modified internucleoside linkage and/or modifiednucleobase.

“Modified oligonucleotide” means an oligonucleotide comprising at leastone modified nucleotide.

“Modified sugar” refers to a substitution or change from a naturalsugar. For example, a 2′-O-methoxyethyl modified sugar is a modifiedsugar.

“Motif” means the pattern of chemically distinct regions in an antisensecompound.

“Statin” means an agent that inhibits the activity of HMG-CoA reductase.

“Symptom of cardiovascular disease or disorder” means a phenomenon thatarises from and accompanies the cardiovascular disease or disorder andserves as an indication of it. For example, angina; chest pain;shortness of breath; palpitations; weakness; dizziness; nausea;sweating; tachycardia; bradycardia; arrhythmia; atrial fibrillation;swelling in the lower extremities; cyanosis; fatigue; fainting; numbnessof the face; numbness of the limbs; claudication or cramping of muscles;bloating of the abdomen; or fever are symptoms of cardiovascular diseaseor disorder.

“Target nucleic acid,” and “target sequence” refer to a nucleic acidcapable of being targeted by a genome editing composition. For example,a target DNA sequence within or adjacent to the ANGPTL3 gene may betargeted by a guide nucleotide associated with a Cas9 nuclease.

Methods for detection and/or measurement of polypeptides in biologicalmaterial are well known in the art and include, but are not limited to,Western-blotting, flow cytometry, ELISAs, RIAs, and various proteomicstechniques. An exemplary method to measure or detect a polypeptide is animmunoassay, such as an ELISA. This type of protein quantitation can bebased on an antibody capable of capturing a specific antigen, and asecond antibody capable of detecting the captured antigen. Exemplaryassays for detection and/or measurement of polypeptides are described inHarlow, E. and Lane, D. Antibodies: A Laboratory Manual, (1988), ColdSpring Harbor Laboratory Press.

Methods for detection and/or measurement of RNA in biological materialare well known in the art and include, but are not limited to,Northern-blotting, RNA protection assay, RT PCR. Suitable methods aredescribed in Molecular Cloning: A Laboratory Manual (Fourth Edition) ByMichael R. Green, Joseph Sambrook, Peter MacCallum 2012, 2,028 pp, ISBN978-1-936113-42-2.

A ribonucleoprotein (RNP) refers to a nucleoprotein that contains RNA. ARNP can be a complex of a ribonucleic acid and an RNA-binding protein.Such a combination can also be referred to as a protein—RNA complex.These complexes can function in a number of biological functions thatinclude, but are not limited to, DNA replication, DNA modification, geneexpression, metabolism and modification of RNA, and pre-mRNA splicing.

The term “nucleobase editors (BEs)” or “base editors (BEs),” as usedherein, refers to a composition, e.g. a fusion protein comprising apolypeptide capable of making a nucleobase modification and a Casprotein. In some embodiments, the fusion protein comprises anuclease-inactive Cas9 (dCas9) fused to a deaminase. In someembodiments, the fusion protein comprises a Cas9 nickase fused to adeaminase. In some embodiments, the fusion protein comprises a D10Xmutation or a H840X mutation of a Cas9 as numbered in a wild type Cas9sequence, e.g. SEQ ID NO: 1, which renders Cas9 capable of cleaving onlyone strand of a nucleic acid duplex. In some embodiments, the baseeditor comprises a programmable DNA nuclease domain fused or linked to adeaminase domain (e.g., adenosine deaminase domain or cytidine deaminasedomain). Details of base editors are described in International PCTApplication Nos. PCT/2017/045381 (WO2018/027078) and PCT/US2016/058344(WO2017/070632), each of which is incorporated herein by reference inits entirety. Also see Komor, A. C., et al., “Programmable editing of atarget base in genomic DNA without double-stranded DNA cleavage” Nature533, 420-424 (2016); Gaudelli, N. M., et al., “Programmable base editingof A•T to G•C in genomic DNA without DNA cleavage” Nature 551, 464-471(2017); Komor, A. C., et al., “Improved base excision repair inhibitionand bacteriophage Mu Gam protein yields C:G-to-T:A base editors withhigher efficiency and product purity” Science Advances 3:eaao4774(2017); Nishida, K. et al. “Targeted nucleotide editing using hybridprokaryotic and vertebrate adaptive immune systems”, Science 353,aaf8729 (2016); Gehrke J M, Cervantes O, Clement M K, Wu Y, Zeng J,Bauer D E, Pinello L, Joung J K. An APOBEC3A-Cas9 base editor withminimized bystander and off-target activities. Nat Biotechnol. 2018November; 36(10):977-982, the entire contents of which are herebyincorporated by reference:

As used herein, the term “biomarker” or “marker” are usedinterchangeably to refer to any biochemical marker, serological marker,genetic marker, or other clinical or echographic characteristic that canbe used to classify a sample from a patient as being associated with anpathological condition, such as a cardiovascular disease or disorder.

As used herein, the term “antibody” includes but is not limited to apopulation of immunoglobulin molecules, which can be polyclonal ormonoclonal and of any class and isotype, or a fragment of animmunoglobulin molecule. There are five major classes ofimmunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these maybe further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3,IgG4, IgA1 (human), IgA2 (human), IgAa (canine), IgAb (canine), IgAc(canine), and IgAd (canine). Such fragment generally comprises theportion of the antibody molecule that specifically binds an antigen. Forexample, a fragment of an immunoglobulin molecule known in the art asFab, Fab′ or F(ab′)2 is included within the meaning of the termantibody.

The term “label,” as used herein, refers to a detectable compound,composition, or solid support, which can be conjugated directly orindirectly (e.g., via covalent or non-covalent means, alone orencapsulated) to a monoclonal antibody or a protein. The label may bedetectable by itself (e.g., radioisotope labels, chemiluminescent dye,electrochemical labels, metal chelates, latex particles, or fluorescentlabels) or, in the case of an enzymatic label, may catalyze chemicalalteration of a substrate compound or composition which is detectable(e.g., enzymes such as horseradish peroxidase, alkaline phosphatase, andthe like). The label employed in the current disclosure could be, but isnot limited to alkaline phosphatase; glucose-6-phosphate dehydrogenase(“G6PDH”); horseradish peroxidase (HRP); chemiluminescers such asisoluminol, fluorescers such as fluorescein and rhodamine compounds;ribozymes; and dyes. The label may also be a specific binding moleculewhich itself may be detectable (e.g., biotin, avidin, streptavidin,digioxigenin, maltose, oligohistidine, e.g., hexa-histidine (SEQ ID NO:114), 2, 4-dinitrobenzene, phenylarsenate, ssDNA, dsDNA, and the like).The utilization of a label produces a signal that may be detected bymeans such as detection of electromagnetic radiation or directvisualization, and that can optionally be measured.

“Substantial binding” or “substantially binding” refer to an amount ofspecific binding or affinity between molecules in an assay mixture underparticular assay conditions. In its broadest aspect, substantial bindingrelates to the difference between a first molecule's incapability ofbinding or recognizing a second molecule, and the first moleculescapability of binding or recognizing a third molecule, such that thedifference is sufficient to allow a meaningful assay to be conducted todistinguish specific binding under a particular set of assay conditions,which includes the relative concentrations of the molecules, and thetime and temperature of an incubation. In another aspect, one moleculeis substantially incapable of binding or recognizing another molecule ina cross-reactivity sense where the first molecule exhibits a reactivityfor a second molecule that is less than 25%, e.g. less than 10%, e.g.,less than 5% of the reactivity exhibited toward a third molecule under aparticular set of assay conditions, which includes the relativeconcentration and incubation of the molecules. Specific binding can betested using a number of widely known methods, e.g, animmunohistochemical assay, an enzyme-linked immunosorbent assay (ELISA),a radioimmunoassay (RIA), or a western blot assay.

As used herein, the term “substantially the same amino acid sequence”includes an amino acid sequence that is similar, but not identical to,the naturally-occurring amino acid sequence. For example, an amino acidsequence, e.g., polypeptide, that has substantially the same amino acidsequence as a flagellin protein can have one or more modifications suchas amino acid additions, deletions, or substitutions relative to theamino acid sequence of the naturally-occurring flagellin protein,provided that the modified polypeptide retains substantially at leastone biological activity of flagellin such as immunoreactivity. The“percentage similarity” between two sequences is a function of thenumber of positions that contain matching residues or conservativeresidues shared by the two sequences divided by the number of comparedpositions times 100. In this regard, conservative residues in a sequenceis a residue that is physically or functionally similar to thecorresponding reference residue, e.g., that has a similar size, shape,electric charge, chemical properties, including the ability to formcovalent or hydrogen bonds, or the like.

The term “targeting moiety” refers to any molecule that provides anenhanced affinity for a selected target, e.g., a cell, cell type,tissue, organ, region of the body, or a compartment, e.g., a cellular,tissue or organ compartment. Some exemplary targeting moieties include,but are not limited to, antibodies, antigens, carbohydrate basemoieties, folates, receptor ligands, carbohydrates, aptamers, integrinreceptor ligands, chemokine receptor ligands, transferrin, biotin,serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDLand HDL ligands. Carbohydrate based targeting moieties include, but arenot limited to, D-galactose, multivalent galactose,N-acetyl-D-galactosamine (GalNAc), multivalent GalNAc, e.g. GalNAc2 andGalNAc3; D-mannose, multivalent mannose, multivalent lactose,N-acetyl-glucosamine, multivalent fucose, glycosylated polyaminoacidsand lectins. The term multivalent indicates that more than onemonosaccharide unit is present. Such monosaccharide subunits can belinked to each other through glycosidic linkages or linked to a scaffoldmolecule.

The term “heterologous” refers to any two or more nucleic acid orpolypeptide sequences that are not normally found in the samerelationship to each other in nature. For instance, a heterologousnucleic acid is typically recombinantly produced, having two or moresequences, e.g., from unrelated genes arranged to make a new functionalnucleic acid, e.g., a promoter from one source and a coding region fromanother source. Similarly, a heterologous polypeptide will often referto two or more subsequences that are not found in the same relationshipto each other in nature (e.g., a fusion protein).

As used herein, the term “fragment” includes a peptide, polypeptide orprotein segment of amino acids of the full-length protein, provided thatthe fragment retains reactivity with at least one antibody in sera ofdisease patients.

An “epitope” is the antigenic determinant on a polypeptide that isrecognized for binding by a paratope on antibodies specific to thepolypeptide, for example, an IBD-associated antibody.

The term “clinical factor” includes a symptom in a patient that isassociated with a cardiovascular disease. Examples of clinical factorsinclude, without limitation, angina; chest pain; shortness of breath;palpitations; weakness; dizziness; nausea; sweating; tachycardia;bradycardia; arrhythmia; atrial fibrillation; swelling in the lowerextremities; cyanosis; fatigue; fainting; numbness of the face; numbnessof the limbs; claudication or cramping of muscles; bloating of theabdomen; or fever. In some embodiments, a diagnosis of a cardiovasculardisease is based upon a combination of analyzing the presence or levelof one or more markers in a patient using statistical algorithms anddetermining whether the patient has one or more clinical factors.

The term “prognosis” includes a prediction of the probable course andoutcome of a pathological condition, for example a cardiovasculardisease, or the likelihood of recovery from the disease. In someembodiments, the use of statistical algorithms provides a prognosis ofcardiovascular disease in a patient. For example, the prognosis can besurgery, development of one or more clinical factors, or recovery fromthe disease.

Provided herein are methods and compositions for targeted delivery oftherapeutic agents such as nucleic acid agents. The therapeutic agentsas used herein may be connected to or associated with a targeting moietyto assist targeted delivery. For example, the therapeutic agent and thetargeting moiety may form a conjugate. The therapeutic agent maycomprise a nucleic acid guided programmable nuclease system complexedwith nucleic acids, such as guide RNAs. In some embodiments, the guideRNAs may be chemically modified. In some embodiments, the modified guideRNAs can be used for the preparation of a medicament for the treatmentof any disease, disorder or condition relating to a gene where the genemay be altered, manipulated, edited, and modified by insertion ordeletion of DNA. According to a further aspect of the disclosure, themodified guide RNA may be used for altering genes by deleting,substituting, repairing or inserting DNA. This can be done inmicroorganisms, or animals, in particular mammals and more particularlyin humans. Human cells or tissue may be genetically altered or amendedusing the guide RNAs of the present disclosure and the CRISPR/Cas systemknown in the art in vitro and then inserted back into the patient inneed thereof. In another aspect of the disclosure there is provided apharmaceutical composition comprising a modified guide RNA according tothe disclosure and a CRISPR-Cas system and a pharmaceutically acceptablecarrier or excipient. The pharmaceutical composition may include avector or a cell with the modified guide RNA of the disclosure. In astill further aspect of the disclosure there is provided a compositioncomprising a modified guide RNA and at least one delivery means selectedfrom GalNAc, polymers, liposomes, peptides, aptamers, antibodies, viralvectors, folate or transferrin.

Nuclease Systems

Provided herein are compositions and methods for targeted delivery ofactive agents, or therapeutic agents, including nucleic acids,polynucleotides or oligonucleotides. The active agent can be apharmaceutic composition, a drug, a polynucleotide, an oligonucleotide,an RNP, a lipid nanoparticle, or a protein-RNA complex. Targeteddelivery as described herein may direct the active agent to a particulardesired location, for example, to specific in vivo positions, cells,tissues, or organs, recognition locations in an intracellular matrix,specific locations within a cell. In some embodiments, the active agentcomprises a guide RNA associated with a nuclease, for example, a CRISPRnuclease. In some embodiments, the active agent comprises a nucleasesystem capable of modifying the activity and/or function of one or moretarget genes, e.g. a PCSK9 or ANGPTL3gene.

In some embodiments, the active agent comprises a genome editingcomposition comprising a nuclease system. In some embodiments, thegenome editing composition is a target specific genome editingcomposition. In some embodiments, the genome editing compositioncomprises a nucleic acid guided programmable nuclease or a portionthereof. In some embodiments of the present disclosure, a nucleasesystem includes at least one nuclease. In some embodiments, the nucleasesystem comprises at least one programmable nuclease. In someembodiments, the nuclease may comprise at least one DNA binding domainand at least one nuclease domain. In some embodiments, the nucleasedomain may be heterologous to the DNA binding domain. In certainembodiments, the nuclease is a DNA endonuclease, and may cleave singleor double-stranded DNA. In certain embodiments, the nuclease may cleaveRNA.

In some embodiments, a nuclease system may include a Cas protein domain(also called a “Cas nuclease”) from a CRISPR/Cas system. The Cas proteinmay comprise at least one domain that interacts with a guide nucleicacid, for example, a guide RNA (gRNA). Additionally, the Cas protein maybe directed to a target sequence by a guide RNA. The guide RNA interactswith the Cas protein as well as the target sequence such that, the Casprotein is directed to the target sequence and may be capable ofcleaving the target sequence. In certain embodiments, e.g., Cas9, theCas protein is a single-protein effector, an RNA-guided nuclease. Insome embodiments, the guide RNA provides the specificity for thetargeted cleavage, and the Cas protein may be universal and paired withdifferent guide RNAs to cleave different target sequences. The terms Casprotein and Cas nuclease are used interchangeably herein.

In some embodiments, the CRISPR/Cas system may comprise Type-I, Type-II,or Type-III system components, or any orthologues thereof. Updatedclassification schemes for CRISPR/Cas loci define Class 1 and Class 2CRISPR/Cas systems, having Types I to V or VI. See, e.g., Makarova etal., Nat Rev Microbiol, 13(11): 722-36 (2015); Shmakov et al., MolecularCell, 60:385-397 (2015). Class 2 CRISPR/Cas systems have single proteineffectors. Cas proteins of Types II, V, and VI may be single-protein,RNA-guided endonucleases, herein called “Class 2 Cas nucleases.” Class 2Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2, and C2c3proteins. Cpf1 protein, Zetsche et al., Cell, 163: 1-13 (2015), ishomologous to Cas9, and contains a RuvC-like nuclease domain. S3.

In some embodiments, the Cas protein may be from a Type-II CRISPR/Cassystem, i.e., a Cas9 protein from a CRISPR/Cas9 system. In someembodiments, the Cas protein may be from a Class 2 CRISPR/Cas system,i.e., a single-protein Cas nuclease such as a Cas9 protein or a Cpf1protein. The Cas9 and Cpf1 family of proteins are enzymes with DNAendonuclease activity, and they can be directed to cleave a desirednucleic acid target by designing an appropriate guide RNA, as describedfurther herein.

A Type-II CRISPR/Cas system component may be from a Type-IIA, Type-IIB,or Type-IIC system. Cas9 nuclease structure and sequences are known tothose skilled in the art (Jinek et al. Science 2012, 337: 816-821;Delcheva et al. Nature 2011, 471: 602-607, incorporated herein byreference). In some embodiments, wild type Cas9 corresponds toStreptococcus pyogenes Cas9 (NCBI Ref No. NC_002737.2, SEQ ID NO: 2) andUniprot Reference Q99ZW2 (SEQ ID NO: 1).

Streptococcus pyogenes Cas9 (wild type) protein sequence (SEQ ID NO: 1)MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLG NTDRHSIKKNLIGALLFDSGEAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLV EEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPD NSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLG LTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPL SASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTE ELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLAR GNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKV KYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDL LKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAFILFDDKVMKQLKRRRYTGWGRLSRKL INGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKG ILQTVKVVDELVKVMGRHKPENIVIEMARENTTKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQN EKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKM KNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKFIVAQILDSRMNTKYDENDK LIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV RKMIAKSEEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSM PVNIVKKTEVTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVK ELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSK YVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD Streptococcus pyogenes Cas9 (wild type)nucleotide sequence (SEQ ID NO: 2) ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCACTGATGA ATATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTT ATAGGGGCTCTTTTATTTGACAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAA GGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGGAGATTTTTTCAAATGAGATGGCGAAAGTAGA TGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCATCCT ATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAACTATCTATCATCTGCGAAAAA AATTGGTAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTT TCGTGGTCATTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTATCCAG TTGGTACAAACCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTGGAGTAGATGCTAAAGCGA TTCTTTCTGCACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAA AAATGGCTTATTTGGGAATCTCATTGCTTTGTCATTGGGTTTGACCCCTAATTTTAAATCAAATTTTGAT TTGGCAGAAGATGCTAAATTACAGCTTTCAAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGC AAATTGGAGATCAATATGCTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGA TATCCTAAGAGTAAATACTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGATTAAACGCTACGATGAA CATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAAAAGTATAAAGAAATCT TTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAA ATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGAT TTGCTGCGCAAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCACTTGGGTGAGCTGCATG CTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAAAAGACAATCGTGAGAAGATTGAAAAAATCTT GACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGG AAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCAT TTATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAAACATAGTTTGCT TTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAAGGAATGCGAAAACCA GCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAACCG TTAAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGA AGATAGATTTAATGCTTCATTAGGTACCTACCATGATTTGCTAAAAATTATTAAAGATAAAGATTTTTTG GATAATGAAGAAAATGAAGATATCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGAGA TGATTGAGGAAAGACTTAAAACATATGCTCACCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCG CCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAA ACAATATTAGATTTTTTGAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATGATA GTTTGACATTTAAAGAAGACATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTTTACATGAACATAT TGCAAATTTAGCTGGTAGCCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAAGTTGTTGATGAATTG GTCAAAGTAATGGGGCGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAATCAGACAACTC AAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCA GATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTCCAA AATGGAAGAGACATGTATGTGGACCAAGAATTAGATATTAATCGTTTAAGTGATTATGATGTCGATCACA TTGTTCCACAAAGTTTCCTTAAAGACGATTCAATAGACAATAAGGTCTTAACGCGTTCTGATAAAAATCG TGGTAAATCGGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGACAACTTCTA AACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGGTTTGAGTGAAC TTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAAT TTTGGATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACC TTAAAATCTAAATTAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATT ACCATCATGCCCATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACT TGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCTAAGTCTGAGCAA GAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAATATCATGAACTTCTTCAAAACAGAAATTA CACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTG GGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAA ACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAAGCTTATTG CTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTCCT AGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACA ATTATGGAAAGAAGTTCCTTTGAAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTA AAAAAGACTTAATCATTAAACTACCTAAATATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCT GGCTAGTGCCGGAGAATTACAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAATTTTTTATAT TTAGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGGAGC AGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAGA TGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAA AATATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTTTGATACAACAA TTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAATCCATCACTGG TCTTTATGAAACACGCATTGATTTGAGTCAGCTAGGAGGTGACTGA

Non-limiting exemplary species that the Cas9 protein or other componentsmay be derived from include Streptococcus pyogenes, Streptococcusthermophilus, Streptococcus sp., Staphylococcus aureus, Listeriainnocua, Lactobacillus gasseri, Francisella novicida, Wolinellasuccinogenes, Sutterella wadsworthensis, Gamma proteobacterium,Neisseria meningitidis, Campylobacter jejuni, Pasteurella multocida,Fibrobacter succinogene, Rhodospirillum rubrum, Nocardiopsisdassonvillei, Streptomyces pristinaespiralis, Streptomycesviridochromogenes, Streptomyces viridochromogenes, Streptosporangiumroseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius,Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacteriumsibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius,Lactobacillus buchneri, Treponema denticola, Microscilla marina,Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonassp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa,Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii,Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridiumbotulinum, Clostridium difficile, Finegoldia magna, Natranaerobiusthermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus,Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobactersp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonashaloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum,Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospiramaxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleuschthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosiphoafricanus, Streptococcus pasteurianus, Neisseria cinerea, Campylobacterlari, Parvibaculum lavamentivorans, Corynebacterium diphtheria, orAcaryochloris marina. In some embodiments, the Cas9 protein may be fromStreptococcus pyogenes. In some embodiments, the Cas9 protein may bederived from Streptococcus thermophilus. In some embodiments, the Cas9protein may be derived from Neisseria meningitidis. In some embodiments,the Cas9 protein may be derived from Staphylococcus aureus.

In some embodiments, a Cas protein may comprise more than one nucleasedomain. For example, a Cas9 protein may comprise at least one RuvC-likenuclease domain (e.g. Cpf1/Cas12a) and at least one HNH-like nucleasedomain (e.g. Cas9). In some embodiments, the Cas9 protein may be capableof introducing a DSB in the target sequence. In some embodiments, theCas9 protein may be modified to contain only one functional nucleasedomain. For example, the Cas9 protein may be modified such that one ofthe nuclease domains is mutated or fully or partially deleted to reduceits nucleic acid cleavage activity. In some embodiments, the Cas9protein may be modified to contain no functional RuvC-like nucleasedomain. In other embodiments, the Cas9 protein may be modified tocontain no functional HNH-like nuclease domain. In some embodiments inwhich only one of the nuclease domains is functional, the Cas9 proteinmay be a nickase that is capable of introducing a single-stranded break(a “nick”) into the target sequence. In some embodiments, a conservedamino acid within a Cas9 protein nuclease domain is substituted toreduce or alter a nuclease activity. In some embodiments, the Casprotein nickase may comprise an amino acid substitution in the RuvC-likenuclease domain. Exemplary amino acid substitutions in the RuvC-likenuclease domain include D10A (based on the S. pyogenes Cas9 protein). Insome embodiments, the nickase may comprise an amino acid substitution inthe HNH-like nuclease domain. Exemplary amino acid substitutions in theHNH-like nuclease domain include E762A, H840A, N863A, H983A, and D986A(based on the S. pyogenes Cas9 protein). In some embodiments, thenuclease system described herein may comprise a nickase and a pair ofguide RNAs that are complementary to the sense and antisense strands ofthe target sequence, respectively. The guide RNAs may direct the nickaseto target and introduce a DSB by generating a nick on opposite strandsof the target sequence (i.e., double nicking). Chimeric Cas9 proteinsmay also be used, where one domain or region of the protein is replacedby a portion of a different protein. For example, a Cas9 nuclease domainmay be replaced with a domain from a different nuclease such as Fok1. ACas9 protein may be a modified nuclease.

Wild type Cas9 and Cas9 sequences from various species may be aligned todetermine corresponding homologous amino acid residues and determineand/or modify amino acid residues at, for example, D10 and H840 of SEQID NO: 1, allowing the generation of Cas9 variants with correspondingmutations of the homologous amino acid residues. The alignment method isknown to those skilled in the art. For example, an alignment may becarried out using the NCBI Constraint-based Multiple Alignment Tool(COBALT, accessible at st-va.ncbi. nlm.nih.gov/tools/cobalt).

In alternative embodiments, the Cas protein may be from a Type-ICRISPR/Cas system. In some embodiments, the Cas protein may be acomponent of the Cascade complex of a Type-I CRISPR/Cas system. Forexample, the Cas protein may be a Cas3 protein. In some embodiments, theCas protein may be from a Type-III CRISPR/Cas system. In someembodiments, the Cas protein may be from a Type-IV CRISPR/Cas system. Insome embodiments, the Cas protein may be from a Type-V CRISPR/Cassystem. In some embodiments, the Cas protein may be from a Type-VICRISPR/Cas system. In some embodiments, the Cas protein may have an RNAcleavage activity.

Fusion Proteins

Provided herein are compositions and methods of targeted modification ofgenes, e.g. PCSK9, ANGPTL3, APOC3, LPA, APOB, MTP, ANGPTL4, ANGPTL8,APOA5, APOE, LDLR, IDOL, NPC1L1, ASGR1, TM6SF2, GALNT2, GCKR, LPL,MLXIPL, SORT1, TRIB1, MARC1, ABCG5, or ABCG8. In certain instances, themodification may be ex vivo or in vivo. In preferred embodiments, thetargeted modification may be directed to a particular type of organ,tissue, or cells, for example, liver hepatocytes. In some embodiments,the target gene is modified genetically with a genome editingcomposition comprising a fusion protein. Accordingly, in someembodiments, provided herein are fusion proteins for targetedmodification of genes. In some embodiments, the fusion protein comprisesa target specific nuclease domain. In some embodiments, the fusionprotein comprises a nucleic acid guided programmable nuclease domain. Insome embodiments, the nucleic acid guided programmable nuclease maycomprise at least one DNA binding domain and at least one nucleasedomain. In some embodiments, the nuclease domain may be heterologous tothe DNA binding domain. In some embodiments, the nuclease domain may bemodified such that the nuclease domain is mutated to reduce its nucleasecleavage activity. In some embodiments, the nuclease activity iscompletely abolished. In some embodiments, the nuclease activity ispartially reduced. In some embodiments, the modified nuclease domain maycomprise a modified Cas protein domain. In certain embodiments, themodified Cas protein domain is a modified Cas9. In some embodiments, themodified Cas9 domain is a nuclease inactive Cas9 (dCas9) domain. In someembodiments, the modified dCas9 domain is a nickase domain. In someembodiments, the modified Cas9 domain contains at least onesubstitutions selected from D10A, N497A, R661A, Q695A, E762A, H840A,N863A, Q926A, H983A and D986A based on the S. pyogenes Cas9 protein. Insome embodiments, the modified nuclease domain is a catalyticallyinactive Cpf1 domain, a catalytically inactive Cas13a domain, acatalytically inactive Cas13b domain, or a catalytically in active Cas13c domain. In some embodiments, the modified nuclease domain is acatalytically inactive CasX, CasY, Cpf1, C2c1, C2c2, C2c3, and Argonauteprotein domain.

In some embodiments, a fusion protein as described herein comprises oneor more functional domains besides the nuclease domain. At least oneprotein domain may be located at the N-terminus, the C-terminus, or inan internal location of the fusion protein. In some embodiments, two ormore heterologous protein domains are at one or more locations on thefusion protein. Non-limiting examples of functional domains include arepressor domain, an activator domain, a methyltransferase domain, ade-methylase domain. In some embodiments, the functional domaincomprises a base-editing enzyme domain. In some embodiments, thefunctional domain is a cytidine deaminase domain. For example, thecytidine deaminase may deaminate a specific cytidine to uracil,resulting in a U-G mismatch which is subsequently resolved via cellularrepair mechanisms to form a U-A base pair, and subsequently a T-A basepair, thereby creating a C-to-T substitution. Cytidine deaminase domainand cytidine-deaminase fusion protein sequences are known to thoseskilled in the art, as described in Komor et al., Science Advances 2017,3(8): eaao4774; Komor et al., Nature 2016, 533: 420-424. In someembodiments, the functional domain is an adenine deaminase domain. Forexample, the adenine deaminase domain may deaminate an adenosine togenerate inosine, which can base pair with cytidine and subsequently becorrected by the cellular repair mechanisms to guanine, therebyconverting A into G. Exemplary adenosine deaminase fusion proteins asdescribed in Gaudelli et al., Nature 2017 551(7681): 464-471, theentirety of which is incorporated herein by reference.

In some embodiments, a fusion protein as described herein comprises anuclear localization signal (NLS). In some embodiments, the fusionprotein may comprise 2, 3, 4, or 5 NLSs. In some embodiments, the fusionprotein may comprise 1-10 NLS(s). The NLS sequence may be fused at the Nterminus and/or the C terminus of the fusion protein. In someembodiments, the NLS may be a monopartite sequence, such as, e.g., theSV40 NLS, PKKKRKV (SEQ ID NO: 3) or PKKKRRV (SEQ ID NO:4). In someembodiments, the NLS may be a bipartite sequence, such as, e.g., the NLSof nucleoplasmin, KRPAATKKAGQAKKKK (SEQ ID NO:5). In some embodiments,the NLS may be genetically modified from its wild-type counterpart. In apreferred embodiment, the fusion protein comprises the sequence ofABE7.10 (SEQ ID NO: 6).

In some embodiments, the fusion protein can further comprise a tagdomain. In some embodiments, the tag domain may comprise a fluorescenttag, a purification tag, an epitope tags, or a reporter gene tag. Insome embodiments, the tag domain may comprise a fluorescent proteindomain. Non-limiting examples of suitable fluorescent proteins includegreen fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, sfGFP,EGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP,ZsGreenl), yellow fluorescent proteins (e.g., YFP, EYFP, Citrine, Venus,YPet, PhiYFP, ZsYellowl), blue fluorescent proteins (e.g., EBFP, EBFP2,Azurite, mKalamal, GFPuv, Sapphire, T-sapphire), cyan fluorescentproteins (e.g., ECFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan), redfluorescent proteins (e.g., mKate, mKate2, mPlum, DsRed monomer,mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem,HcRedl, AsRed2, eqFP611, mRasberry, mStrawberry, Jred), and orangefluorescent proteins (mOrange, mKO, Kusabira-Orange, MonomericKusabira-Orange, mTangerine, tdTomato) or any other suitable fluorescentprotein. In some embodiments, the tag domain may comprise a purificationtag and/or an epitope tag. Non-limiting exemplary tags includeglutathione-S-transferase (GST), chitin binding protein (CBP), maltosebinding protein (MBP), thioredoxin (TRX), poly(NANP), tandem affinitypurification (TAP) tag, myc, AcV5, AU1, AUS, E, ECS, E2, FLAG, HA, nus,Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G,6×His (SEQ ID NO: 114), biotin carboxyl carrier protein (BCCP), andcalmodulin. In some embodiments, the tag domain may comprise a reportergene domain. Non-limiting exemplary reporter genes includeglutathione-S-transferase (GST), horseradish peroxidase (HRP),chloramphenicol acetyltransferase (CAT), beta-galactosidase,beta-glucuronidase, luciferase, or fluorescent proteins.

In additional embodiments, the nuclease in the nuclease systems maycomprise one or more programmable nucleases other than a Cas protein.For example, the nuclease may be chosen from a meganuclease (e.g.,homing endonucleases), ZFN, TALEN, and megaTAL.

Naturally-occurring meganucleases may recognize and cleavedouble-stranded DNA sequences of about 12 to 40 base pairs, and arecommonly grouped into five families. In some embodiments, themeganuclease may be chosen from the LAGLIDADG family, the GIY-YIGfamily, the HNH family, the His-Cys box family, and the PD-(D/E)XKfamily. In some embodiments, the DNA binding domain of the meganucleasemay be engineered to recognize and bind to a sequence other than itscognate target sequence. In some embodiments, the DNA binding domain ofthe meganuclease may be fused to a heterologous nuclease domain. In someembodiments, the meganuclease, such as a homing endonuclease, may befused to TAL modules to create a hybrid protein, such as a “megaTAL”protein. The megaTAL protein may have improved DNA targeting specificityby recognizing the target sequences of both the DNA binding domain ofthe meganuclease and the TAL modules.

ZFNs are fusion proteins comprising a zinc-finger DNA binding domain(“zinc fingers” or “ZFs”) and a nuclease domain. Eachnaturally-occurring ZF may bind to three consecutive base pairs (a DNAtriplet), and ZF repeats are combined to recognize a DNA target sequenceand provide sufficient affinity. Thus, engineered ZF repeats may becombined to recognize longer DNA sequences, such as, e.g., 9-, 12-, 15-,or 18-bp, etc. In some embodiments, the ZFN may comprise ZFs fused to anuclease domain from a restriction endonuclease. For example, therestriction endonuclease may be FokI. In some embodiments, the nucleasedomain may comprise a dimerization domain, such as when the nucleasedimerizes to be active, and a pair of ZFNs comprising the ZF repeats andthe nuclease domain may be designed for targeting a target sequence,which comprises two half target sequences recognized by each ZF repeatson opposite strands of the DNA molecule, with an interconnectingsequence in between (which is sometimes called a spacer in theliterature). For example, the interconnecting sequence may be 5 to 7 bpin length. When both ZFNs of the pair bind, the nuclease domain maydimerize and introduce a DSB within the interconnecting sequence. Insome embodiments, the dimerization domain of the nuclease domain maycomprise a knob-into-hole motif to promote dimerization. For example,the ZFN may comprise a knob-into-hole motif in the dimerization domainof FokI.

The DNA binding domain of TALENs usually comprises a variable number of34 or 35 amino acid repeats (“modules” or “TAL modules”), with eachmodule binding to a single DNA base pair, A, T, G, or C. Adjacentresidues at positions 12 and 13 (the “repeat-variable di-residue” orRVD) of each module specify the single DNA base pair that the modulebinds to. Though modules used to recognize G may also have affinity forA, TALENs benefit from a simple code of recognition—one module for eachof the 4 bases—which greatly simplifies the customization of aDNA-binding domain recognizing a specific target sequence. In someembodiments, the TALEN may comprise a nuclease domain from a restrictionendonuclease. For example, the restriction endonuclease may be FokI. Insome embodiments, the nuclease domain may dimerize to be active, and apair of TALENS may be designed for targeting a target sequence, whichcomprises two half target sequences recognized by each DNA bindingdomain on opposite strands of the DNA molecule, with an interconnectingsequence in between. For example, each half target sequence may be inthe range of 10 to 20 bp, and the interconnecting sequence may be 12 to19 bp in length. When both TALENs of the pair bind, the nuclease domainmay dimerize and introduce a DSB within the interconnecting sequence. Insome embodiments, the dimerization domain of the nuclease domain maycomprise a knob-into-hole motif to promote dimerization. For example,the TALEN may comprise a knob-into-hole motif in the dimerization domainof FokI.

Certain embodiments of the disclosure also provide nucleic acidsencoding the nuclease system described herein provided on a vector. Insome embodiments, the nucleic acid may be a DNA molecule. In otherembodiments, the nucleic acid may be an RNA molecule. In someembodiments, the nucleic acid encoding the nuclease may be an mRNAmolecule.

In some embodiments, the nucleic acid encoding the nuclease may be codonoptimized for efficient expression in one or more eukaryotic cell types.In some embodiments, the nucleic acid encoding the nuclease may be codonoptimized for efficient expression in one or more mammalian cells. Insome embodiments, the nucleic acid encoding the nuclease may be codonoptimized for efficient expression in human cells. Methods of codonoptimization including codon usage tables and codon optimizationalgorithms are available in the art.

Guide Polynucleotides

In some embodiments of the present disclosure, a CRISPR/Cas nucleasesystem includes at least one guide polynucleotide, for example, a guideRNA. In some embodiments, the guide RNA and the Cas protein may form aribonucleoprotein (RNP), e.g., a CRISPR/Cas complex. The guide RNA mayguide the Cas protein to a target sequence on a target nucleic acidmolecule, where the guide RNA hybridizes with and the Cas proteincleaves the target sequence. In some embodiments, the CRISPR/Cas complexmay be a Cpf1/guide RNA complex. In some embodiments, the CRISPR complexmay be a Type-II CRISPR/Cas9 complex. In some embodiments, the Casprotein may be a Cas9 protein. In some embodiments, the CRISPR/Cas9complex may be a Cas9/guide RNA complex.

A guide nucleic acid (e.g., guide RNA) can bind to a Cas protein andtarget the Cas protein to a specific location within a targetpolynucleotide. A guide nucleic acid can comprise a nucleicacid-targeting segment and a Cas protein binding segment.

A guide nucleic acid can refer to a nucleic acid that can hybridize toanother nucleic acid, for example, the target polynucleotide in thegenome of a cell. A guide nucleic acid can be RNA, for example, a guideRNA. A guide nucleic acid can be DNA. A guide nucleic acid can compriseDNA and RNA. A guide nucleic acid can be single stranded. A guidenucleic acid can be double-stranded. A guide nucleic acid can comprise anucleotide analog. A guide nucleic acid can comprise a modifiednucleotide. The guide nucleic acid can be programmed or designed to bindto a sequence of nucleic acid site-specifically.

A guide nucleic acid can comprise one or more modifications to providethe nucleic acid with a new or enhanced feature. A guide nucleic acidcan comprise a nucleic acid affinity tag. A guide nucleic acid cancomprise synthetic nucleotide, synthetic nucleotide analog, nucleotidederivatives, and/or modified nucleotides.

The guide nucleic acid can comprise a nucleic acid-targeting region(e.g., a spacer region), for example, at or near the 5′ end or 3′ end,that is complementary to a protospacer sequence in a targetpolynucleotide. The spacer of a guide nucleic acid can interact with aprotospacer in a sequence-specific manner via hybridization (basepairing). The protospacer sequence can be located 5′ or 3′ ofprotospacer adjacent motif (PAM) in the target polynucleotide. Thenucleotide sequence of a spacer region can vary and determines thelocation within the target nucleic acid with which the guide nucleicacid can interact. The spacer region of a guide nucleic acid can bedesigned or modified to hybridize to any desired sequence within atarget nucleic acid.

A guide nucleic acid can comprise two separate nucleic acid molecules,which can be referred to as a double guide nucleic acid. A guide nucleicacid can comprise a single nucleic acid molecule, which can be referredto as a single guide nucleic acid (e.g., sgRNA). In some embodiments,the guide nucleic acid is a single guide nucleic acid comprising a fusedCRISPR RNA (crRNA) and a transactivating crRNA (tracrRNA). In someembodiments, the guide nucleic acid is a single guide nucleic acidcomprising a crRNA. In some embodiments, the guide nucleic acid is asingle guide nucleic acid comprising a crRNA but lacking a tracRNA. Insome embodiments, the guide nucleic acid is a double guide nucleic acidcomprising non-fused crRNA and tracrRNA. An exemplary double guidenucleic acid can comprise a crRNA-like molecule and a tracrRNA-likemolecule. An exemplary single guide nucleic acid can comprise acrRNA-like molecule. An exemplary single guide nucleic acid can comprisea fused crRNA-like and tracrRNA-like molecules.

A crRNA can comprise the nucleic acid-targeting segment (e.g., spacerregion) of the guide nucleic acid and a stretch of nucleotides that canform one half of a double-stranded duplex of the Cas protein-bindingsegment of the guide nucleic acid.

A tracrRNA can comprise a stretch of nucleotides that forms the otherhalf of the double-stranded duplex of the Cas protein-binding segment ofthe gRNA. A stretch of nucleotides of a crRNA can be complementary toand hybridize with a stretch of nucleotides of a tracrRNA to form thedouble-stranded duplex of the Cas protein-binding domain of the guidenucleic acid.

The crRNA and tracrRNA can hybridize to form a guide nucleic acid. ThecrRNA can also provide a single-stranded nucleic acid targeting segment(e.g., a spacer region) that hybridizes to a target nucleic acidrecognition sequence (e.g., protospacer). The sequence of a crRNA,including spacer region, or tracrRNA molecule can be designed to bespecific to the species in which the guide nucleic acid is to be used.

A guide RNA for a CRISPR/Cas9 system typically comprises a CRISPR RNA(crRNA) and a tracr RNA (tracr). A guide RNA for a CRISPR/Cpf1 systemtypically comprises a crRNA. In some embodiments, the crRNA may comprisea targeting sequence that is complementary to and hybridizes with thetarget sequence on the target nucleic acid molecule. The crRNA may alsocomprise a sequence that is complementary to and hybridizes with aportion of the tracrRNA. In some embodiments, the crRNA may parallel thestructure of a naturally occurring crRNA transcribed from a CRISPR locusof a bacteria, where the targeting sequence acts as the spacer of theCRISPR/Cas9 system.

The guide RNA may target any sequence of interest via the targetingsequence of the crRNA. In some embodiments, the degree ofcomplementarity between the targeting sequence of the guide RNA and thetarget sequence on the target nucleic acid molecule may be about 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In someembodiments, the targeting sequence of the guide RNA and the targetsequence on the target nucleic acid molecule may be 100% complementary.In other embodiments, the targeting sequence of the guide RNA and thetarget sequence on the target nucleic acid molecule may contain at leastone mismatch. For example, the targeting sequence of the guide RNA andthe target sequence on the target nucleic acid molecule may contain 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches. In some embodiments, thetargeting sequence of the guide RNA and the target sequence on thetarget nucleic acid molecule may contain 1-6 mismatches. In someembodiments, the targeting sequence of the guide RNA and the targetsequence on the target nucleic acid molecule may contain 5 or 6mismatches.

The length of the targeting sequence may depend on the CRISPR/Cas9system and components used. For example, different Cas9 proteins fromdifferent bacterial species have varying optimal targeting sequencelengths. Accordingly, the targeting sequence may comprise 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 35, 40, 45, 50, or more than 50 nucleotides in length. Insome embodiments, the targeting sequence may comprise 18-30 nucleotidesin length. In some embodiments, the targeting sequence may comprise19-24 nucleotides in length. In some embodiments, the targeting sequencemay comprise 20 nucleotides in length.

The crRNA and the tracr may comprise any sequence with sufficientcomplementarity to promote the formation of a functional CRISPR/Cas9complex. In some embodiments, the complementary sequence between thecrRNA and the tracr may comprise all or a portion of the sequence (alsocalled a “tag” or “handle”) of a naturally-occurring crRNA that iscomplementary to the tracr RNA in the same CRISPR/Cas9 system. In someembodiments, the complementary sequence may comprise all or a portion ofa repeat sequence from a naturally-occurring CRISPR/Cas9 system. In someembodiments, the complementary sequence may comprise a truncated ormodified tag or handle sequence. In some embodiments, the degree ofcomplementarity between the tracr RNA and the portion of thecomplementary portion that hybridizes with the tracr RNA along thelength of the shorter of the two sequences may be about 40%, 50%, 60%,70%, 80%, or higher, but lower than 100%. In some embodiments, the tracrRNA and the portion that hybridizes with the tracr RNA are not 100%complementary along the length of the shorter of the two sequencesbecause of the presence of one or more bulge structures on the tracrand/or wobble base pairing. The length of the tracr RNA complementaryportion to tracr may depend on the CRISPR/Cas9 system or the tracr RNAused. For example, the complementary portion may comprise 10-50nucleotides, or more than 50 nucleotides in length. In some embodiments,the complementary portion may comprise 15-40 nucleotides in length. Inother embodiments, the complementary portion may comprise 20-30nucleotides in length. In yet other embodiments, the complementaryportion may comprise 22 nucleotides in length. When a dual guide RNA isused, for example, the length of the complementary portion may have noupper limit.

In some embodiments, the tracr RNA may comprise all or a portion of awild-type tracr RNA sequence from a naturally-occurring CRISPR/Cas9system. In some embodiments, the tracr RNA may comprise a truncated ormodified variant of the wild-type tracr RNA. The length of the tracr RNAmay depend on the CRISPR/Cas9 system used. In some embodiments, thetracr RNA may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100nucleotides in length. In certain embodiments, the tracr is at least 26nucleotides in length. In additional embodiments, the tracr is at least40 nucleotides in length. In some embodiments, the tracr RNA maycomprise certain secondary structures, such as, e.g., one or morehairpins or stem-loop structures, or one or more bulge structures.

In some embodiments, the guide RNA may comprise two RNA molecules and isreferred to herein as a “dual guide RNA” or “dgRNA”. In someembodiments, the dgRNA may comprise a first RNA molecule comprising acrRNA, and a second RNA molecule comprising a tracr RNA. The first andsecond RNA molecules may form a RNA duplex via the base pairing betweenthe flagpole on the crRNA and the tracr RNA.

In some embodiments, the guide RNA may comprise a single RNA moleculeand is referred to herein as a “single guide RNA” or “sgRNA”. In someembodiments, the sgRNA may comprise a crRNA covalently linked to a tracrRNA. In some embodiments, the crRNA and the tracr RNA may be covalentlylinked via a linker. In some embodiments, the single-molecule guide RNAmay comprise a stem-loop structure via the base pairing between theflagpole on the crRNA and the tracr RNA.

Certain embodiments of the disclosure also provide nucleic acids, e.g.,vectors, encoding the guide RNA described herein. In some embodiments,the nucleic acid may be a DNA molecule. In other embodiments, thenucleic acid may be an RNA molecule. In some embodiments, the nucleicacid may comprise a nucleotide sequence encoding a crRNA. In someembodiments, the nucleotide sequence encoding the crRNA comprises atargeting sequence flanked by all or a portion of a repeat sequence froma naturally-occurring CRISPR/Cas system. In some embodiments, thenucleic acid may comprise a nucleotide sequence encoding a tracr RNA. Insome embodiments, the crRNA and the tracr RNA may be encoded by twoseparate nucleic acids. In some embodiments, the crRNA and the tracr RNAmay be encoded by a single nucleic acid. In some embodiments, the crRNAand the tracr RNA may be encoded by opposite strands of a single nucleicacid. In other embodiments, the crRNA and the tracr RNA may be encodedby the same strand of a single nucleic acid.

In certain embodiments, more than one guide RNA can be used with aCRISPR/Cas nuclease system. Each guide RNA may contain a differenttargeting sequence, such that the CRISPR/Cas system cleaves more thanone target sequence. In some embodiments, one or more guide RNAs mayhave the same or differing properties such as activity or stabilitywithin the Cas9 RNP complex. Where more than one guide RNA is used, eachguide RNA can be encoded on the same or on different vectors. Thepromoters used to drive expression of the more than one guide RNA may bethe same or different.

The methods of selecting guide RNAs for efficient targeting with highspecificity and low off-target effect are known to those skilled in theart. For programmable base-editing, [selection of a genomic sequencecontaining a target sequence may be as described in Komor et al, Nature,533, 420-424 (2016) is incorporated herein by reference. The guide RNAsequence and PAM preference define the genomic target sequence(s) ofprogrammable nuclease domains (e.g. Cas9, dCas9, Cas9n, Cpf1, NgAgodomains). Methods of reducing off-target binding as described in Hsu etal (Nature biotechnology, 2013, 31(9):827-832), Fusi et al (bioRxiv021568; doi: http://dx.doi.org/10.1101/021568), Chari et al (NatureMethods, 2015, 12(9):823-6), Doench et al (Nature Biotechnology, 2014,32(12): 1262-7), Wang et al (Science, 2014, 343(6166): 80-4),Moreno-Mateos et al (Nature Methods, 2015, 12(10):982-8), Housden et al(Science Signaling, 2015, 8(393):r59), Haeussler et al, (Genome Biol.2016; 17: 148) are incorporated herein by reference. The potential forthe formation of bulges between the guide RNA and the target DNA andother parameters that may influence target sequence binding may also beconsidered as described in Bae et al (Bioinformatics, 2014, 30, 1473-5)Housden et al (Science Signaling, 2015, 8(393):r59), and Farboud et al(Genetics, 2015, 199(4):959-71) are also incorporated herein byreference.

RNA Modification

Provided herein are modified RNA molecules suitable for targeted ex vivoand in vivo delivery systems. A modified RNA molecule may comprise twoor more linked ribonucleic acid subunits. Non-limiting exemplarymodified RNAs include CRISPR guide RNA, short interfering RNA (siRNA),microRNA (miRNA), short hairpin RNA (shRNA), small nuclear RNA (snRNA),messenger RNA (mRNA), precursor mRNA (pre-mRNA), antisense RNA (asRNA),and heteronuclear RNA (hnRNA). Modified RNAs as described hereinencompass both the RNA sequence and any structural embodiment thereof,e.g. single stranded, double stranded, triple stranded, circular,helical, hairpin, stem loop, buldge, etc. A modified RNA may comprise alength of at least about 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100,200, 300, 400, or 500 bases. A modified RNA may comprise a length of atleast about 1 kilobase (kb), 2 kb, 3 kb, 4 kb, 5 kb, 10 kb, 20 kb, 50kb, or more. In some embodiments, the modified RNA is a CRISPR guide RNA(gRNA). A gRNA may be a single guide RNA or a dual guide RNA. In someembodiments, the modified RNA is a mRNA. In some embodiments, a mRNA canbe isolated from a cell or a tissue. In some embodiments, a mRNA can betranscribed from a DNA. In some embodiments, a mRNA can be chemicallysynthesized.

In certain embodiments, modified RNA molecules provided herein areresistant to degradation by RNases or other exonucleases. In certainembodiments, modified RNA molecules provided herein are stabilized toprevent degradation by endonucleases. In some embodiments, modified RNAmolecules provided herein are suitable for in vivo delivery and inducesless cellular immune receptor activation (e.g. TLR, RIG-I) as comparedto unmodified RNA. RNA modifications as described in Diebold (2008) AdvDrug Deliv Rev. Apr. 29; 60(7):813-23) and Sorrentino (1998) Cell MolLife Sci. August; 54(8):785-94, the entirety of both are incorporatedherein by reference.

In addition to “unmodified” or “natural” nucleobases such as the purinenucleobases adenine (A) and guanine (G), and the pyrimidine nucleobasesthymine (T), cytosine (C) and uracil (U), many modified nucleobases ornucleobase mimetics known to those skilled in the art are amenable withthe compounds described herein. The unmodified or natural nucleobasescan be modified or replaced to provide oligonucleotides having improvedproperties. For example, nuclease resistant oligonucleotides can beprepared with these bases or with synthetic and natural nucleobases(e.g., inosine, xanthine, hypoxanthine, nebularine, isoguanisine, ortubercidine) and any one of the oligomer modifications described herein.Alternatively, substituted or modified analogs of any of the above basesand “universal bases” can be employed. When a natural base is replacedby a non-natural and/or universal base, the nucleotide is said tocomprise a modified nucleobase and/or a nucleobase modification herein.Modified nucleobase and/or nucleobase modifications also includenatural, non-natural and universal bases, which comprise conjugatedmoieties, e.g. a ligand described herein. Preferred conjugate moietiesfor conjugation with nucleobases include cationic amino groups which canbe conjugated to the nucleobase via an appropriate alkyl, alkenyl or alinker with an amide linkage.

As used herein, “unmodified” or “natural” nucleobases include the purinebases adenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Exemplary modified nucleobases include, butare not limited to, other synthetic and natural nucleobases such asinosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidine,2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine,2-(aminoalkyll)adenine, 2-(aminopropyl)adenine,2-(methylthio)-N6-(isopentenyl)adenine, 6-(alkyl)adenine,6-(methyl)adenine, 7-(deaza)adenine, 8-(alkenyl)adenine,8-(alkyl)adenine, 8-(alkynyl)adenine, 8-(amino)adenine, 8-(halo)adenine,8-(hydroxyl)adenine, 8-(thioalkyl)adenine, 8-(thiol)adenine,N6-(isopentyl)adenine, N6-(methyl)adenine, N6, N6-(dimethyl)adenine,2-(alkyl)guanine, 2-(propyl)guanine, 6-(alkyl)guanine,6-(methyl)guanine, 7-(alkyl)guanine, 7-(methyl)guanine,7-(deaza)guanine, 8-(alkyl)guanine, 8-(alkenyl)guanine,8-(alkynyl)guanine, 8-(amino)guanine, 8-(halo)guanine,8-(hydroxyl)guanine, 8-(thioalkyl)guanine, 8-(thiol)guanine,N-(methyl)guanine, 2-(thio)cytosine, 3-(deaza)-5-(aza)cytosine,3-(alkyl)cytosine, 3-(methyl)cytosine, 5-(alkyl)cytosine,5-(alkynyl)cytosine, 5-(halo)cytosine, 5-(methyl)cytosine,5-(propynyl)cytosine, 5-(propynyl)cytosine, 5-(trifluoromethyl)cytosine,6-(azo)cytosine, N4-(acetyl)cytosine, 3-(3-amino-3-carboxypropyl)uracil,2-(thio)uracil, 5-(methyl)-2-(thio)uracil,5-(methylaminomethyl)-2-(thio)uracil, 4-(thio)uracil,5-(methyl)-4-(thio)uracil, 5-(methylaminomethyl)-4-(thio)uracil,5-(methyl)-2,4-(dithio)uracil, 5-(methylaminomethyl)-2,4-(dithio)uracil,5-(2-aminopropyl)uracil, 5-(alkyl)uracil, 5-(alkynyl)uracil,5-(allylamino)uracil, 5-(aminoallyl)uracil, 5-(aminoalkyl)uracil,5-(guanidiniumalkyl)uracil, 5-(1,3-diazole-1-alkyl)uracil,5-(cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil,5-(dimethylaminoalkyl)uracil, 5-(halo)uracil, 5-(methoxy)uracil,uracil-5-oxyacetic acid, 5-(methoxycarbonylmethyl)-2-(thio)uracil,5-(methoxycarbonyl-methyl)uracil, 5-(propynyl)uracil,5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil,dihydrouracil, N-(methyl)uracil, 5-uracil (i.e., pseudouracil),2-(thio)pseudouracil, 4-(thio)pseudouracil, 2,4-(dithio)psuedouracil,5-(alkyl)pseudouracil, 5-(methyl)pseudouracil,5-(alkyl)-2-(thio)pseudouracil, 5-(methyl)-2-(thio)pseudouracil,5-(alkyl)-4-(thio)pseudouracil, 5-(methyl)-4-(thio)pseudouracil,5-(alkyl)-2,4-(dithio)pseudouracil, 5-(methyl)-2,4-(dithio)pseudouracil,1-methylpseudouracil (N1-methylpseudouracil), 1-substitutedpseudouracil, 1-substituted 2(thio)-pseudouracil, 1-substituted4-(thio)pseudouracil, 1-substituted 2,4-(dithio)pseudouracil,1-(aminocarbonylethylenyl)-pseudouracil,1-(aminocarbonylethylenyl)-2(thio)-pseudouracil,1-(aminocarbonylethylenyl)-4-(thio)pseudouracil, 1-(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil,1-(aminoalkylaminocarbonylethylenyl)-pseudouracil,1-(aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil,1-(aminoalkylaminocarbonylethylenyl)-4-(thio)pseudouracil,1-(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil,1,3-(diaza)-2-(oxo)-phenoxazin-1-yl,1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl,1,3-(diaza)-2-(oxo)-phenthiazin-1-yl,1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-substituted1,3-(diaza)-2-(oxo)-phenoxazin-1-yl,7-substituted-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-substituted1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-substituted1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl,7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl,7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl,7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl,7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl,7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl,7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl,7-(guanidiniumalkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl,7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl,1,3,5-(triaza)-2,6-(dioxa)-naphthalene, inosine, xanthine, hypoxanthine,nubularine, tubercidine, isoguanisine, inosinyl, 2-aza-inosinyl,7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl,nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl,3-(methyl)isocarbostyrilyl, 5-(methyl)isocarbostyrilyl,3-(methyl)-7-(propynyl)isocarbostyrilyl, 7-(aza)indolyl,6-(methyl)-7-(aza)indolyl, imidizopyridinyl,9-(methyl)-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl,7-(propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl,2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl,phenyl, napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl,tetracenyl, pentacenyl, difluorotolyl,4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole,6-(azo)thymine, 2-pyridinone, 5-nitroindole, 3-nitropyrrole,6-(aza)pyrimidine, 2-(amino)purine, 2,6-(diamino)purine, 5-substitutedpyrimidines, N2-substituted purines, N6-substituted purines,06-substituted purines, substituted 1,2,4-triazoles,pyrrolo-pyrimidin-2-on-3-yl, 6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,para-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,ori/zo-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,bis-ori/zo-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,ori/zo-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,bis-ori/zo-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl,2-oxo-pyridopyrimidine-3-yl, or any O-alkylated or N-alkylatedderivatives thereof. Alternatively, substituted or modified analogs ofany of the above bases and “universal bases” can be employed. Auniversal nucleobase is any nucleobase that can base pair with all ofthe four naturally occurring nucleobases without substantially affectingthe melting behavior, recognition by intracellular enzymes or activityof the oligonucleotide duplex. Some exemplary universal nucleobasesinclude, but are not limited to, 2,4-difluorotoluene, nitropyrrolyl,nitroindolyl, 8-aza-7-deazaadenine, 4-fluoro-6-methylbenzimidazle,4-methylbenzimidazle, 3-methyl isocarbostyrilyl, 5-methylisocarbostyrilyl, 3-methyl-7-propynyl isocarbostyrilyl, 7-azaindolyl,6-methyl-7-azaindolyl, imidizopyridinyl, 9-methyl-imidizopyridinyl,pyrrolopyrizinyl, isocarbostyrilyl, 7-propynyl isocarbostyrilyl,propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4-methylinolyl,4,6-dimethylindolyl, phenyl, napthalenyl, anthracenyl, phenanthracenyl,pyrenyl, stilbenyl, tetracenyl, pentacenyl, and structural derivativesthereof (see for example, Loakes, 2001, Nucleic Acids Research, 29,2437-2447, incorporated herein by reference in its entirety). Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808; thosedisclosed in International Application No. PCT U.S. Ser. No. 09/038,425,filed Mar. 26, 2009; those disclosed in the Concise Encyclopedia OfPolymer Science And Engineering, pages 858-859, Kroschwitz, J. I, ed.John Wiley & Sons, 1990; those disclosed by English et al, AngewandteChemie, International Edition, 1991, 30, 613; those disclosed inModified Nucleosides in Biochemistry, Biotechnology and Medicine,Herdewijin, P. Ed. Wiley-VCH, 2008; and those disclosed by Sanghvi, Y.S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke,S. T. and Lebleu, B., Eds., CRC Press, 1993. Contents of all of theabove are herein incorporated by reference.

In some embodiments, modified RNAs as described herein are modified toattach a delivery and/or targeting moiety such as GalNAc. Suitably,GalNAc can be attached to the 3′-end, 5 ‘-end of the RNA, or both. Insome embodiments, GalNAc is attached to the 3’-end. In some embodiments,the modified RNAs show improvements relative to their unmodifiedequivalents. Such improvements can relate to improved specificity (suchthat, for example, off-target effects are reduced or a lowerconcentration of gRNA is required), improved stability (e.g. resistanceto enzymes such as nucleases), improved functionality or decreasedimmunogenicity or immunostimulatory properties. In some embodiments, themodified RNAs show efficient transfection into cells and/or improvedproperties allowing it to be delivered and maintained in an organism,tissue, body fluid or cell such that the RNA, e.g. a guide RNA,functionality can take place. Methods for measuring these improvedproperties compared to their unmodified equivalents are known to thoseskilled in the art and include those methods described herein.Accordingly in some embodiments, provided herein is a modified RNA thathas increased stability compared to the unmodified equivalent. Byun-modified equivalent is meant a RNA, e.g. a guide RNA which targetsthe same specific gene sequence and interacts with the same Cas9 orCRISPR nuclease and comprises natural nucleotides. Increased stabilityincludes increased stability or resistance to enzymes such as nucleaseswhich may be present in cells, tissues or body fluids and which mayotherwise contribute to degradation of the RNA such that is hasdecreased functionality. In certain embodiments, increased stabilityincludes increased serum stability. In some embodiments, provided hereinis a modified guide RNA that has increased CRISPR activity compared tothe un-modified equivalent. Methods for measuring CRISPR activity aredescribed herein. In some embodiments, provided herein is a modifiedguide RNA that has decreased immunostimulatory activity compared to theun-modified equivalent. Methods for measuring immunostimulation aredescribed herein.

Provided herein are modified mRNA molecules for targeted delivery. Forexample, a mRNA that encodes a CRISPR enzyme, e.g. a Cas9, Cas12b, or abase editor (BE) may be modified for specific tissue targeting. The mRNAmay be modified at least one nucleotide at the 2′ position and/orbackbone modification. In some embodiments, the nucleotides in a mRNAcan include modification of the thioates. In some embodiments, a mRNAcan include modification of one or more of 2′-OMe, 2′-F,N-1-methyl-psuedouridine, 5-methyluridine 5-methoxyuridine, and5-ethoxyuridine.

In certain embodiments, mRNA sequences provided herein comprise a fullymodified or partially modified mRNA. In some embodiments, a mRNAcomprises chemical modifications in a fragment, or multiple fragments ofthe entire length. Non-limiting exemplary modifications and modificationpatterns of the nucleotides of a mRNA, or segments thereof, are shown inTable 2 and Table 3.

Provided herein are modified guide RNAs for use with CRISPR/Cas systemwhere the guide RNA may be modified by a chemical modification of atleast one nucleotide at the 2′ position and/or backbone modification.The backbone modification can include modification of the thioates. Incertain embodiments, the nucleotides that are modified are selected froma group of nucleotides which interact with the Cas amino acids in theCas protein to effect binding of the guide RNA to Cas. In certainembodiments, the modification can comprise that the 2′-OH on thenucleotide is replaced with at least one of H, —OR, —R,—O—C₁-C₆-alkylene-OR, —O—C₁-C₆-alkylene-OH, halo, —SH, —SR, —NH₂, —NHR,—N(R)₂, —C₁-C₆-alkylene-NH₂, —C₁-C₆-alkylene-NHR, —C₁-C₆-alkylene-N(R)₂,or CN, wherein each R is independently C₁-C₆ alkyl, C₂-C₆ alkenyl, orC₂-C₆ alkynyl and halo is F, Cl, Br or I. In some instances, themodifications are 2′-O-methyl and/or 2′-F. In some embodiments, themodification comprises one or more of 2′-F, phosphorothioateinternucleotide linkage modification, acyclic nucleotides, LNA, HNA,CeNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy,2′-O—N-methylacetamido (2′-O-NMA), a 2′-O-dimethylaminoethoxyethyl(2′-0-DMAEOE), 2′-0-aminopropyl (2′-O-AP), and 2′-ara-F modification. Insome embodiments, the modification comprises 2′-MOE. In someembodiments, the modification comprises phosphorothioate internucleotidelinkage modification. In some embodiments, the modification comprises4-O-alkyl ribosugars such as 4′-methoxy and 4′-ethoxy modifications.

Suitably, the modified guide RNA can be applied with the S. pyogenesCRISPR/Cas9 system, or any other CRISPR/Cas systems such as those inStaphylococcus aureus or Staphylococcus haemolyticus. The modification,or similar modification patterns, can also be made to guide RNAs forCpf1 from Lachnospiraceae bacterium ND2006 or Cpf1 from Acidominococcusspecies BV3L6.

In certain embodiments, guide RNA sequences comprise a fully modifiedsingle guide RNA. In some embodiments, a guide RNA comprises chemicalmodifications in the tracr RNA portion. Non-limiting exemplarymodifications and modification patterns of the nucleotides of a guideRNA according to the disclosure are shown in Table 2 and Table 3.

Modified guide RNAs as described herein may be used in complex withCRISPR/Cas system or CRISPR/Cas enzymes to effect alteration in a targetgene or DNA sequence. The CRISPR/Cas enzymes may comprise CRISPRnucleases, such as Cas9, Cpf1, C2c1, C2c2, or C2c3. In some embodiments,the CRISPR/Cas enzyme may comprise CRISPR nucleases with modified orreduced nuclease activity, such as a nuclease inactive Cas9 or Cpf1. Forexample, mutations may be introduced into one or both nucleasesubdomains of a Cas9 enzyme to generate a Cas9 nickase or a nucleaseinactive Cas9. Exemplary inactivating mutations in Cas9 includealterations at positions D10, E762, H840, N854, N863, or D986 of SEQ IDNO: 1. For example, a D10Amutation in the RuvC subdomain and an H840Amutation in the HNH subdomain of Cas9 renders the Cas9 nucleaseinactive. A D10A mutation in the RuvC subdomain or a H840A in the HNHsubdomain of Cas9 generates a Cas9 nickase. Additional amino acidsubstitutions in Cas9 are discussed in WO15/89354, which is incorporatedherein in its entirety.

The modified guide RNAs share sequence identity with, or is capable ofhybridize to, a target nucleotide such as a target gene or a target DNAsequence. In some embodiments, modified guide RNA has at least 100%,99%, 98%, 96%, 95%, 90%, 85%, 80%, 75%, or 70% correspondence oridentity to a target nucleotide of a gene or target DNA.

The nucleotides as described herein can be synthetic or chemicallymodified. For example, guide RNAs provided herein can be synthetic orchemically modified guide RNAs. The nucleotides in the guide RNA thatare modified may be those corresponding to one or more nucleotides inthe binding region of the guide RNA with Cas9 and/or the nucleotides inthe binding region of the guide RNA with the target DNA. Remainingunmodified nucleotides of the guide RNA may be those required to beidentified for minimal binding of Cas9 to the 2′-OH location on thebases. In some embodiments, the nucleotides may be modified at the 2′position of the sugar moiety of the nucleotide. In some embodiments, the2′—OH group of the sugar moiety is replaced by a group selected from H,OR, R, halo, SH, SR, H2, NHR, N(R)2 or CN, wherein R is C₁-C₆ alkyl,alkenyl or alkynyl and halo is F, Cl, Br or I. Other modifications mayinclude, inverted (deoxy) abasics, amino, fluoro, chloro, bromo, CN, CF,methoxy, imidazole, carboxylate, thioate, CI to CIO lower alkyl,substituted lower alkyl, alkaryl or aralkyl, heterozycloalkyl;heterozycloalkaryl; aminoalkylamino; polyalkylamino or substitutedsilyl. Methods for making RNAs with specific sequences and modificationsare known by those skilled in the art, for example, in Dellinger et al.(201 1), J. Am. Chem. Soc, 133, 11540; U.S. Pat. No. 8,202,983; Kumar etal., (2007), J. Am. Chem. Soc, 129, 6859-64; WO2013176844, the entiretyof which are incorporated herein by reference.

In some embodiments, polynucleotides or oligonucleotides as providedherein may be synthetic. For example, guide RNAs maybe chemicallysynthesized guide RNAs. Synthetic RNA production yield is based onsequences and modifications. 2′-O— methyl modifications have been shownto increase coupling efficacy or efficiency during RNA synthesis andtherefore increase yield of chemically synthesized RNA. Furthermore,nucleotides may be modified by phosphorothioates. Phosphothioate(phosphorothioate)(PS) bonds substitute a sulphur atom for anon-bridging oxygen in the phosphate backbone of an oligonucleotide.Accordingly, exemplary nucleotides of the disclosure include, but arenot limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs),threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptidenucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having aβ-D-ribo configuration, a-LNA having an a-L-ribo configuration (adiastereomer of LNA), 2′-amino-LNA having a 2′-amino functionalization,and 2′-amino-a-LNA having a 2′-amino functionalization) or hybridsthereof.

Conjugates for Targeted Delivery

Provided herein are conjugates suitable for targeted delivery of agents,such as mRNA, guide RNA, miRNA, siRNA, DNA, peptides, or other micro ormacro molecules. A conjugate can contain one or more aptamers, ligands,or moieties for targeted delivery ex vivo or in vivo. In someembodiments, a conjugate comprises a targeting moiety (or ligand), alinker, and an active agent (or payload) that is connected to thetargeting moiety. An active agent can be a therapeutic agent, aprophylactic agent, or a diagnostic/prognostic agent. An active agentmay have a capability of manipulating a physiological function (e.g.,gene expression) in a subject. An active agent maybe a guide RNA, amRNA, a miRNA, a siRNA, a DNA, or a peptide. The active agent may beconnected with the targeting moiety via a linker, via a non-covalentlinkage, via nucleobase paring, or any combination thereof. In someembodiments, the conjugate may be a conjugate between a single activeagent and a single targeting moiety with the formula (I): X—Y—Z, whereinX is the targeting moiety; Y is a linker; and Z is the guide RNA. Incertain embodiments, one targeting ligand can be conjugated to two ormore active agents, wherein the conjugate has the formula: X—(Y—Z)_(n).For example, the conjugate may comprise a guide RNA and a mRNA. Incertain embodiments, one active agent can be linked to two or moretargeting ligands wherein the conjugate has the formula: (X—Y)_(n)—Z. Inother embodiments, one or more targeting moieties may be connected toone or more active pay loads wherein the conjugate formula may be(X—Y—Z)_(n). In various combinations, the formula of the conjugatesmaybe, for example, X—Y—Z—Y—X, (X—Y—Z)_(n)—Y—Z, or X—Y—(X—Y—Z)_(n),wherein X is a targeting moiety; Y is a linker; Z is an active agent,e.g. a guide RNA. The number of each moiety in the conjugate may varydependent on types of agents, sizes of the conjugate, delivery targets,particles used to packaging the conjugate, other active agents (e.g.,immunologic adjuvants) and routes of administration. Each occurrence ofX, Y, and Z can be the same or different, e.g. the conjugate can containmore than one type of targeting moiety, more than one type of linker,and/or more than one type of active agent, n is an integer equal to orgreater than 1. In some embodiments, n is an integer between 1 and 50,or between 2 and 20, or between 5 and 40. In some embodiments, n may bean integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 41, 43, 44, 45, 46, 47, 48, 49 or 50.

In some embodiments, an active agent, e.g., a guide RNA may be deliveredto cells and tissues using viral, polymeric and liposomal formulations,cell-penetrating peptides, aptamers, ligands, or conjugates and antibodyapproaches. A moiety or ligand may direct guide RNAs to particularorgan, tissue, or cell, for example, a liver hepatocyte, and may bereferred to as a targeting moiety. In some embodiments, targetingmoieties modify one or more properties of the attached molecule (e.g., amRNA or a guide RNA), including but not limited to pharmacodynamic,pharmacokinetic, binding, absorption, cellular distribution, cellularuptake, charge and clearance.

Exemplary moieties that can be attached to a herein described activeagent include, but are not limited to, intercalators, reportermolecules, polyamines, polyamides, polyethylene glycols, thioethers,polyethers, cholesterols, thiocholesterols, cholic acid moieties,folate, lipids, phospholipids, biotin, phenazine, phenanthridine,anthraquinone, adamantane, acridine, fluoresceins, rhodamines,coumarins, dyes, lipid moieties such as a cholesterol moiety (Letsingeret al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553); cholic acid(Manoharan et al, Bioorg. Med. Chem. Lett., 1994, 4, 1053); a thioether,e.g., hexyl-S-tritylthiol (Manoharan et al, Ann. NY. Acad. Sci., 1992,660, 306; Manoharan et al, Bioorg. Med. Chem. Let., 1993, 3, 2765); athiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533); analiphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaraset al, EMBO J., 1991, 10, 111; Kabanov et al, FEBS Lett., 1990, 259,327; Svinarchuk et al, Biochimie, 1993, 75, 49); a phospholipid, e.g.,di-hexadecyl-rac-glycerol ortriethylammonium-1,2-di-0-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al, Tetrahedron Lett., 1995, 36, 3651; Shea et al, Nucl.Acids Res., 1990, 18, 3777); a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969); adamantaneacetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651); apalmityl moiety (Mishra et al, Biochim. Biophys. Acta, 1995, 1264, 229);or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety(Crooke et al, J. Pharmacol. Exp. Ther., 1996, 277, 923), all referencesincorporated herein in their entirety. Targeting moieties may includenaturally occurring molecules, or recombinant or synthetic molecules,including, but not limited to, GalNAc or derivative thereof (e.g., adimer, trimer, or tetramer of GalNAc or derivative thereof), polylysine(PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acidanhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinylether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamidecopolymer (HMPA), polyethylene glycol (PEG, e.g., PEG-2K, PEG-5K,PEG-10K, PEG-12K, PEG-15K, PEG-20K, PEG-40K), MPEG, [MPEG]2, polyvinylalcohol (PVA), polyurethane, poly(2-ethylacryllic acid),N-isopropylacrylamide polymers, polyphosphazine, polyethylenimine,cationic groups, spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, mucin, glycosylated polyaminoacids,transferrin, bisphosphonate, polyglutamate, polyaspartate, aptamer,asialofetuin, hyaluronan, procollagen, immunoglobulins (e.g.,antibodies), insulin, transferrin, albumin, sugar-albumin conjugates,intercalating agents (e.g., acri dines), cross-linkers (e.g. psoralen,mitomycin C), porphyrins (e.g., TPPC4, texaphyrin, Sapphyrin),polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine),artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g,steroids, bile acids, cholesterol, cholic acid, adamantane acetic acid,1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-0(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, 03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine), peptides (e.g., an alpha helicalpeptide, amphipathic peptide, RGD peptide, cell permeation peptide,endosomolytic/fusogenic peptide), alkylating agents, phosphate, amino,mercapto, polyamino, alkyl, substituted alkyl, radiolabeled markers,enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g.,naproxen, aspirin, vitamin E, folic acid), synthetic ribonucleases(e.g., imidazole, bisimidazole, histamine, imidazole clusters,acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles),dinitrophenyl, HRP, AP, antibodies, hormones and hormone receptors,lectins, carbohydrates, multivalent carbohydrates, vitamins (e.g.,vitamin A, vitamin E, vitamin K, vitamin B, e.g., folic acid, B12,riboflavin, biotin and pyridoxal), vitamin cofactors,lipopolysaccharide, an activator of p38 MAP kinase, an activator ofNF-κB, taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine,myoservin, tumor necrosis factor alpha (TNFalpha), interleukin-1 beta,gamma interferon, natural or recombinant low density lipoprotein (LDL),natural or recombinant high-density lipoprotein (HDL), and acell-permeation agent (e.g., a.helical cell-permeation agent), peptideand peptidomimetic ligands, including those having naturally occurringor modified peptides, e.g., D or L peptides; a, (3, or y peptides;N-methyl peptides; azapeptides; peptides having one or more amide, i.e.,peptide, linkages replaced with one or more urea, thiourea, carbamate,or sulfonyl urea linkages; or cyclic peptides; amphipathic peptidesincluding, but not limited to, cecropins, lycotoxins, paradaxins,buforin, CPF, bombinin-like peptide (BLP), cathelicidins, ceratotoxins,S. clava peptides, hagfish intestinal antimicrobial peptides (HFIAPs),magainines, brevinins-2, dermaseptins, melittins, pleurocidin, H2Apeptides, Xenopus peptides, esculentinis-1, and caerins. Apeptidomimetic (also referred to herein as an oligopeptidomimetic) is amolecule capable of folding into a defined three-dimensional structuresimilar to a natural peptide. The peptide or peptidomimetic ligand ormoiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20,25, 30, 35, 40, 45, or 50 amino acids long. In some embodiments, thetargeting moiety may be other peptides such as somatostatin, octeotide,LHRH (luteinizing hormone releasing hormone), epidermal growth factorreceptor (EGFR) binding peptide, aptide or bipodal peptide,RGD-containing peptides, a protein scaffold such as a fibronectindomain, a single domain antibody, a stable scFv, or other homingpeptides. As non-limiting examples, a protein or peptide based targetingmoiety may be a protein such as thrombospondin, tumor necrosis factors(TNF), annexin V, an interferon, angiostatin, endostatin, cytokine,transferrin, GM-CSF (granulocyte-macrophage colony-stimulating factor),or growth factors such as vascular endothelial growth factor (VEGF),hepatocyte growth factor (HGF), (platelet-derived growth factor (PDGF),basic fibroblast growth factor (bFGF), and epidermal growth factor(EGF). In some embodiments, the targeting moiety maybe an antibody, anantibody fragment, RGD peptide, folic acid or prostate specific membraneantigen (PSMA). In some embodiments, the protein scaffold may be anantibody-derived protein scaffold. Non-limiting examples include singledomain antibody (dAbs), nanobody, single-chain variable fragment (scFv),antigen-binding fragment (Fab), Avibody, minibody, CH₂D domain, Fcab,and bispecific T-cell engager (BiTE) molecules. In some embodiments,scFv is a stable scFv, wherein the scFv has hyperstable properties. Insome embodiments, the nanobody may be derived from the single variabledomain (VHH) of camelidae antibody.

In some embodiments, a targeting moiety recognizes or binds a targetcell, a marker, or a molecule that is present exclusively orpredominantly on the surface of particular cells. For example, atargeting moiety may bind a tumor antigen and direct the activatingagent, e.g. a guide RNA-Cas complex to a malignant cell. In someembodiments, the targeting moiety recognizes an intra-cellular protein.In some embodiments, a targeting moiety directs a conjugate to specifictissues, cells, or locations in a cell. The targeting moiety can directthe conjugate in culture or in a whole organism, or both. In each case,the targeting moiety may bind to a receptor that is present on thesurface of or within the targeted cell(s), wherein the targeting moietybinds to the receptor with an effective specificity, affinity andavidity. In other embodiments the targeting moiety targets the conjugateto a specific tissue such as the liver, kidney, lung or pancreas. Inother cases, targeting moieties can direct the conjugate to cells of thereticular endothelial or lymphatic system, or to professional phagocyticcells such as macrophages or eosinophils. In some embodiments, thetargeting moiety may recognize a RTK receptor, an EGF receptor, a serineor threonine kinase, G-protein coupled receptor, methyl CpG bindingprotein, cell surface glycoprotein, cancer stem cell antigen or marker,carbonic anhydrase, cytolytic T lymphocyte antigen, DNAmethyltransferase, an ectoenzyme, aglycosylphosphatidylinositol-anchored co-receptor, a glypican-relatedintegral membrane proteoglycan, a heat shock protein, a hypoxia inducedprotein, a multi drug resistant transporter, a Tumor-associatedmacrophage marker, a tumor associated carbohydrate antigen, a TNFreceptor family member, a transmembrane protein, a tumor necrosis factorreceptor superfamily member, a tumour differentiation antigen, a zincdependent metallo-exopeptidase, a zinc transporter, a sodium-dependenttransmembrane transport protein, a member of the SIGLEC family oflectins, or a matrix metalloproteinase.

In some embodiments, a herein described conjugate, e.g., a guide RNAconjugate, comprise at least one N-Acetyl-Galactosamine (GalNAc),N—Ac-Glucosamine (GluNAc), or mannose (e.g., mannose-6-phosphate). Insome embodiments, a targeting moiety comprise at least oneN-Acetyl-Galactosamine (GalNAc), N—Ac-Glucosamine (GluNAc), or mannose(e.g., mannose-6-phosphate).

In some embodiments, a herein described conjugate comprises one or moretargeting moieties that comprise N-acetylgalactosamine (GalNAc) orGalNAc derivatives. Such a conjugate is also referred to herein as aGalNAc conjugate. In some embodiments, the conjugate targets a RNA to aparticular cell, e.g., a liver cell, e.g., a hepatocyte. In someembodiments, the GalNAc derivatives can be attached via a linker, e.g.,a bivalent or trivalent branched linker.

In some embodiments, a herein described conjugate is a carbohydrateconjugate. In some embodiments, a carbohydrate conjugate comprises amonosaccharide. In some embodiments, the monosaccharide is anN-acetylgalactosamine (GalNAc). GalNAc and GalNAc derivatives arecapable of binding Asialoglycoprotein receptor (ASGPR), also known asAshwell-Morell receptor, a lectin predominantly expressed on liverhepatocytes.

GalNAc conjugates are described, for example, in U.S. Pat. No.8,106,022, the entire content of which is hereby incorporated herein byreference. In some embodiments, the GalNAc conjugate serves as a ligandthat targets the guide RNA to particular cells. In some embodiments, theGalNAc conjugate targets the guide RNA to liver cells, e.g., by servingas a ligand for the asialoglycoprotein receptor of liver cells (e.g.,hepatocytes). In some embodiments, the carbohydrate conjugate comprisesone or more GalNAc derivatives. The GalNAc derivatives may be attachedvia a linker, e.g., a bivalent or trivalent branched linker. In someembodiments the GalNAc conjugate is conjugated to the 3′ end of thesense strand. In some embodiments, the GalNAc ligand is conjugated tothe active agent (e.g., to the 3′ end of guide RNA) via a linker, e.g.,a linker as described herein. In some other embodiments, the GalNAcligand is conjugated to the active agent (e.g., to the 5′ end of guideRNA) via a linker, e.g., a linker as described herein.

In some embodiments, the GalNAc ligand may be conjugated to a shortmeroligonucleotide via a linker and spacer, wherein the shorteroligonucleotide conjugate is complementary to a segment of an RNA. TheRNA encompasses all length, structure, and forms of RNA moledules,including, for example, a mRNA of interest and guide RNA of interest. Insome embodiments, a shortmer —GalNAc conjugate and a RNA constitute apharmaceutical composition. For example, a shortmer GalNAc-conjugatedoligonucleotide and a RNA, e.g. a coupling sequence, together mayconstitute a pharmaceutical composition via W—C H-bonding ofcomplementary nucleotides. The shortmer oligonucleotide conjugate maycomprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more than 50nucleotides in length. In some embodiments, the coupling sequence maycomprise 15-40 nucleotides in length. In some embodiments, the couplingsequence may comprise 19-30 nucleotides in length. In some embodiments,the coupling sequence may comprise 20-24 nucleotides in length.

In some embodiments, provided herein are pharmaceutical compositionscomprising one or more GalNAc conjugated shortmer oligonucleoitdes andone or more RNAs. In some embodiments, a single GalNAc conjugatedshortmer oligonucleotide, e.g., a GalNAc conjugated RNA, may becomplementary to multiple oligonucleotide segments within a RNA. Forexample, the single GalNAc conjugated shortmer may comprise a couplingsequence complementary to multiple segments within a RNA. In someembodiments, multiple GalNAc ligand conjugated shortmer oligonucleotidesthat are complementary to multiple oligonucleotide segments within anRNA may constitute a pharmaceutical composition.

In certain embodiments, the targeting moiety of a herein describedconjugate comprises a ligand having a structure shown in Table 1 below.

TABLE 1 Non-limiting examples of targeting moiety structures.

(1-1a) and (1-1b)

(1-2a) and (1-2b)

(1-3a) and (1-3b)

(1-4a) and (1-4b)

(1-5a) and (1-5b)

(1-6a) and (1-6b)

(1-7a) and (1-7b)

(1-8a) and (1-8b)

(1-9a) and (1-9b)

(1-10A) and (1-10b)

(1-11a) and (1-11b)

(1-12a) and (1-12b)

(1-13a) and (1-13b)

(1-14a) and (1-14b)

(1-15a) and (1-15b)

(1-16a) and (1-16b)

(1-17)

(1-18)

(1-19)

(1-20)

(1-21a) and (1-21b)

(1-22a) and (1-22b)

(1-23a) and (1-23b)

(1-24a) and (1-24b)

(1-25a) and (1-25b)

(1-26A) and (1-26b)

(1-27a) and (1-27b)

(1-28a) and (1-28b)

As shown in Table 1, each of t, n, p, q and m is independently 0, or aninteger from 1 to 30. In some embodiments, each of t, n, p, q and m ofTable 1 is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14,13, 15, 16, 17, 18, 19, or 20. In some embodiments, each of t, n, p, qand m of Table 1 is independently 0, 1, 2, 3, 4, or 5. In someembodiments, each of t, n, p, q and m of Table 1 is independently 0, 1,2, or 3. In some embodiments, each of t, n, p, q and m of Table 1 isindependently 1 or 2. Accordingly, it should be understood that it iscontemplated herein that in some embodiments of compounds of Table 1, tis 0 to 10. In some embodiments, t is 1 to 5. In some embodiments, t is10 to 20. In some embodiments, t is 1 or 2. In some embodiments, t is 1.In some embodiments, t is 2. In some embodiments of compounds of Table1, m is 0 to 10. In some embodiments, m is 1 to 5. In some embodiments,m is 10 to 20. In some embodiments, m is 1 or 2. In some embodiments, mis 1. In some embodiments, m is 2. In some embodiments of compounds ofTable 1, n is 0 to 10. In some embodiments, n is 1 to 5. In someembodiments, n is 10 to 20. In some embodiments, n is 1 or 2. In someembodiments, n is 1. In some embodiments, n is 2. In some embodiments ofcompounds of Table 1, p is 0 to 10. In some embodiments, p is 1 to 5. Insome embodiments, p is 10 to 20. In some embodiments, p is 1 or 2. Insome embodiments, p is 1. In some embodiments, p is 2. In someembodiments of compounds of Table 1, q is 0 to 10. In some embodiments,q is 1 to 5. In some embodiments, q is 10 to 20. In some embodiments, qis 1 or 2. In some embodiments, q is 1. In some embodiments, q is 2. Insome embodiments, each R is OH or NHC(O)CH₃ or combination thereof. Insome embodiments, x is 0 or an integer from 1-5 in compound (1-1a),(1-2a), (1-3a), (1-4a), (1-5a), (1-6a), (1-7a), (1-8a), (1-9a), (1-10a),(1-11a)), (1-12a), (1-16a), (1-21a), (1-22a), (1-23a), (1-24a), (1-25a)and (1-26a) of Table 1. In some embodiments, x is 0 or an integer from1-5 in compound (1-1b), (1-2b), (1-3b), (1-4b), (1-5b), (1-6b), (1-7b),(1-8b), (1-9b), (1-10b), (1-11b)), (1-12b), (1-16b), (1-21b), (1-22b),(1-23b), (1-24b), (1-25b) and (1-26b) of Table 1. In some embodiments, xis 1. In some embodiments, x is 2. In some embodiments, x is 0. In someembodiments, x is 3. In some embodiments, x is 4. In some embodiments, xis 5.

Targeting moieties can be conjugated to nucleobases, sugar moieties, orinternucleosidic linkages of a nucleic acid, e.g. a guide RNA or mRNA.Conjugation to purine nucleobases or derivatives thereof can occur atany position including, endocyclic and exocyclic atoms. In someembodiments, the 2-, 6-, 7-, or 8-positions of a purine nucleobase areattached to a moiety. Conjugation to pyrimidine nucleobases orderivatives thereof can also occur at any position. In some embodiments,the 2-, 5-, and 6-positions of a pyrimidine nucleobase can besubstituted with a moiety. When a moiety is conjugated to a nucleobase,the preferred position is one that does not interfere withhybridization, i.e., does not interfere with the hydrogen bondinginteractions needed for base pairing.

Conjugation to sugar moieties of nucleosides can occur at any carbonatom. Example carbon atoms of a sugar moiety that can be attached to aconjugate moiety include the 2′, 3′, and 5′ carbon atoms. Thegamma-position can also be attached to a conjugate moiety, such as in anabasic residue. Internucleosidic linkages can also bear conjugatemoieties. For phosphorus-containing linkages (e.g., phosphodiester,phosphorothioate, phosphorodithiotate, phosphoroamidate, and the like),the conjugate moiety can be attached directly to the phosphorus atom orto an O, N, or S atom bound to the phosphorus atom. For amine- oramide-containing internucleosidic linkages (e.g., PNA), the conjugatemoiety can be attached to the nitrogen atom of the amine or amide or toan adjacent carbon atom.

There are numerous methods for preparing conjugates of oligonucleotides.Generally, an oligonucleotide is attached to a conjugate moiety bycontacting a reactive group (e.g., OH, SH, amine, carboxyl, aldehyde,and the like) on the oligonucleotide with a reactive group on theconjugate moiety. In some embodiments, one reactive group iselectrophilic and the other is nucleophilic. For example, anelectrophilic group can be a carbonyl-containing functionality and anucleophilic group can be an amine or thiol. Methods for conjugation ofnucleic acids and related oligomeric compounds with and without linkinggroups are well described in the literature such as, for example, inManoharan in Antisense Research and Applications, Crooke and LeBleu,eds., CRC Press, Boca Raton, Fla., 1993, Chapter 17, which isincorporated herein by reference in its entirety.

A targeting moiety can be attached to an active agent or therapeuticnucleic acid described herein, such as a guide RNA, via RNA-RNA orRNA-DNA base pairing and hybridization. Not intended to be bound by anytheories, a targeting moiety can comprise a coupling sequence that iscapable of recognizing or binding an active agent, e.g., a guide RNA ora mRNA. In some embodiments, a targeting moiety comprises a couplingsequence capable of hybridizing to a 5′ portion, a 3′ portion, or amiddle portion of a guide RNA. A guide RNA that hybridizes with acoupling sequence may comprise an extension. For example, the couplingsequence may be able to hybridize with the extension sequence of theguide RNA, thereby directing the guide RNA to desired in vivo, ex vivo,intercellular or intracellular locations, while the guide RNAfunctionality such as interaction with CRISPR enzyme or binding withtarget sequence(s) is not affected. In some embodiments, the guide RNAcomprises an extension that includes a polynucleotide tail. In someembodiments, the guide nucleic acid comprises a poly(A) tail, a poly(U)tail, or a poly(T) tail capable of hybridizing with a poly(U) tail, apoly(A) tail, or a poly(A) tail of the coupling sequence respectively.In some embodiments, the guide nucleic acid may be a guide RNA thatcomprises the sequence of (A)n or (U)n. In some embodiments, the guidenucleic acid may comprise DNA and may comprise the sequence of (A)n or(T)n. In some embodiments, the coupling sequence may comprise thesequence of (A)n (SEQ ID NO: 115), (U)n (SEQ ID NO: 116) or (T)n (SEQ IDNO: 117). As instantly used, n may be any integer between 1 and 200.

A coupling sequence may share sequence identity or complementarity witha nucleic acid active agent, or a portion thereof. In some embodiments,a coupling sequence may share at least 10%, at least 15%, at least 20%,at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100% of identity with a guide RNA describedherein, or a portion of such guide RNA. In some embodiments, a couplingsequence may share at least 10%, at least 15%, at least 20%, at least25%, at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least98%, at least 99%, or 100% of identity with the complementary sequenceof a guide RNA described herein, or the complementary of a portion ofsuch guide RNA. In some embodiments, the coupling sequence may compriseidentity or complementarity with at least 2, at least 3, at least 4, atleast 5, at least 6, at least 7, at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, at least 20, at least 21, atleast 22, at least 23, at least 24, at least 25, at least 26, at least27, at least 28, at least 29, at least 30, at least 31, at least 32, atleast 33, at least 34, at least 35, at least 36, at least 37, at least38, at least 39, at least 40, at least 41, at least 42, at least 43, atleast 44, at least 45, at least 46, at least 47, at least 48, at least49, at least 50, at least 51, at least 52, at least 53, at least 54, atleast 55, at least 56, at least 57, at least 58, at least 59, at least60, at least 61, at least 62, at least 63, at least 64, at least 65, atleast 66, at least 67, at least 68, at least 69, at least 70, at least71, at least 72, at least 73, at least 74, at least 75, at least 76, atleast 77, at least 78, at least 79, at least 80, at least 81, at least82, at least 83, at least 84, at least 85, at least 86, at least 87, atleast 88, at least 89, at least 90, at least 91, at least 92, at least93, at least 94, at least 95, at least 96, at least 97, at least 98, atleast 99, or at least 100 contiguous nucleobases of the guide RNA or acomplementary thereof.

In some embodiments, a targeting moiety may comprise or be associatedwith a coupling sequence that is chemically modified. In someembodiments, the coupling sequence comprises an extension thathybridizes with a therapeutic nucleic acid, e.g. a guide RNA, or aportion thereof. In some embodiments, the extension of the couplingsequence may be chemically modified. In some embodiments, thetherapeutic nucleic acid, e.g. a guide RNA, may comprise an extension.In some embodiments, the extension of the guide RNA may be chemicallymodified. Non-limiting examples of guide RNA extensions andcomplementary or substantially complementary coupling sequenceextensions are shown below in Table 2.

TABLE 2 Exemplary RNA GaINAc conjugate single chemicalentity coupling sequences. RNA- RNA-GalNAc conjugate GalNAcsingle chemical SEQ Conjugate entity coupling ID No. sequences No 2-15′-RNA-AAAAAAAAA 7 AAAA 3′-ususuuuuuuuu 8 uuus(GalNAc)-5′ 2-25′-RNA-AAAAAAAAA 9 AAAA 3′-(GalNAc)uuuu 10 uuuuuuususu 2-35′-RNA-AAAAAAAA 11 AAAAA 3′-(GalNAc)usus 12 uuuuuuuuuuu(Gal NAc) 2-45′-RNA-AAAAAAAAA 13 AAAAAAAAAAAAAAA 3′-ususuuuuuuuu 14 uuuuuuuuuuuuuus(GalNAc)-5′ 2-5 5′-RNA-AAAAAAAAA 15 AAAAAAAAAAAAAAA 3′-(GalNAc)uuuu 16uuuuuuuuuuuuuuu uuususu 2-6 5′-RNA-AAAAAAAAA 17 AAAAAAAAAAAAAAA3′-(GalNAc)uuuu 18 uuuuuuuuuuuuuuu uuuuus(GalNAc)- 5′ 2-75′-RNA-AAAAAAAA 19 AAAAA 3′-usUsuUuUuUuU 20 uUus(GalNAc)-5′ 2-85′-RNA-AAAAAAAAA 21 AAAA 3-(GalNAc)uUuUu 22 UuUuUusUsu 2-95′-RNA-AAAAAAAAA 23 AAAA 3′-(GalNAc)usUs 24 uUuUuUuUuUu(Gal NAc) 2-105′-RNA-AAAAAAAAA 25 AAAAAAAAAAAAAAA 3′-usUsuUuUuUuU 26 uUuUuUuUuUuUuus(GalNAc)-5′ 2-11 5′-RNA-AAAAAAAA 27 AAAAAAAAAAAAAAA A 3′-(GalNAc)uUuU 28uUuUuUuUuUuUuUu UuUsusu 2-12 5′-RNA-AAAAAAAA 29 AAAAAAAAAAAAAAA A3′-(GalNAc)uUuU 30 uUuUuUuUuUuUuUu UuUuus(GalNAc)- 5′ 2-135′-RNA-AAAAAAAAA 31 AAAA 3′-UsUsUUUUUUUU 32 UUUs(GalNAc)-5′ 2-145′-RNA-AAAAAAAAA 33 AAAA 3′-(GalNAc)UUUU 34 UUUUUUUsUsU 2-155′-RNA-AAAAAAAA 35 AAAAA 3′-(GalNAc)UsUs 36 UUUUUUUUUUU(Gal NAc) 2-165′-RNA-AAAAAAAA 37 AAAAAAAAAAAAAAA A 3′-UsUsUUUUUUUU 38 UUUUUUUUUUUUUUs(GalNAc)-5′ 2-17 5′-RNA-AAAAAAAAA 39 AAAAAAAAAAAAAAA 3′-(GalNAc)UUUU 40UUUUUUUUUUUUUUU UUUsLJsU 2-18 5′-RNA-AAAAAAAAA 41 AAAAAAAAAAAAAAA3′-(GalNAc)UUUU 42 UUUUUUUUUUUUUUU UUUUUs(GalNAc)- 5′ 2-195′-RNA-AAAAAAAAA 43 AAAA 3′-usTsuTuTuTuT 44 uTus(GalNAc)-5′ 2-205′-RNA-AAAAAAAA 45 AAAAA 3′-(GalNAc)uTuT 46 uTuTuTusTsu 2-215′-RNA-AAAAAAAAA 47 AAAA 3′-(GalNAc)usTs 48 uTuTuTuTuTu(Gal NAc) 2-225′-RNA-AAAAAAAA 49 AAAAAAAAAAAAAAA A 3′-usTsuTuTuTuT 50 uTuTuTuTuTuTuus(GalNAc)-5′ 2-23 5′-RNA-AAAAAAAAA 51 AAAAAAAAAAAAAAA 3′-(GalNAc)uTuTu 52TuTuTuTuTuTuTuT uTsusu 2-24 5′-RNA-AAAAAAAAA 53 AAAAAAAAAAAAAAA3′-(GalNAc)uTuT 54 uTuTuTuTuTuTuTu TuTuus(GalNAc)- 5′ 2-255′-RNA-AAAAAAAAA 55 AAAA 3′-TsTsTTTTTTTT 56 TTTs(GalNAc)-5′- 2-265′-RNA-AAAAAAAA 57 AAAAA 3′-(GalNAc)TTTT 58 TTTTTTusTsT 2-275′-RNA-AAAAAAAAA 59 AAAA 3′-(GalNAc)TsTs 60 TTTTTTTTTTT(Gal NAc) 2-285′-RNA-AAAAAAAAA 61 AAAAAAAAAAAAAAA 3-TsTsTTTTTTTTT 62 TTTTTTTTTTTTTs(GalNAc)-5′ 2-29 5′-RNA-AAAAAAAAA 63 AAAAAAAAAAAAAAA 3′-(GalNAc)TTTT 64TTTTTTTTTTTTTTT TTTsTsT 2-30 5′-RNA-AAAAAAAA 65 AAAAAAAAAAAAAAA A3′-(GalNAc)TTTT 66 TTTTTTTTTTTTTTT TTTTTs(GalNAc)- 5′

As used in Table 2, uppercase A, C, G and U refer to ribonucleotidesbearing nucleobases adenine, cytosine, guanidine and uracil,respectively; lowercase a, c, g and u refer to modified (e.g., 2′-OMe or2′-MOE) ribonucleotides bearing nucleobases adenine, cytosine, guanidineand uracil, respectively; letter “T” refers to thymidine ordeoxythymidine; and letter “s” refers to a phosphorus-containing linkage(such as a phosphorothioate (PS) linkage, a phosphodiester linkage, or aphosphorodithioate linkage). As used in Table 2, “(GalNAc)” refers to atargeting moiety such as one comprising a GalNAc or a derivativethereof. As used in Table 2, “(GalNAc)” also encompasses a targetingmoiety that comprises multiple GalNAc structures or derivatives thereofsuch as a dimer, trimer, a tetramer of GalNAc or derivatives thereof,including the GalNAc structures described in Table 1. In someembodiments, “s” represents a phosphorothioate (PS) linkages. Asdisclosed herein, the nucleotide sequences and modification patternsencompass all length, structure, and type of RNAs or fragments thereof,CRISPR guide RNAs, e.g. sgRNAs, dual guide RNAs, or mRNAs. For example,nucleotide sequences and modification patterns as described in Table 2above may indicate RNA sequences and modification patterns in a singleguide RNA, a dual guide RNA, a nuclease mRNA, or any fragment or segmentthereof.

Non-limiting examples of guide RNAs conjugated to receptor targetingmoeity and coupling sequences comprising a targeting moiety are providedin Table 3 below. The (GalNAc) conjugate moiety is covalently conjugatedto the 3′ and/or 5′ end of the guide RNA and/or covalently conjugated tothe 3′ and/or 5′ end of the guide RNA with additional nucleotidespacer(s) between the ligand and guide RNA. The guide RNA conjugates 3-1and 3-2 (Table 3) are representative examples of direct conjugation ofthe GalNAc ligand to the guide RNA. The guide RNA conjugates 3-10 to3-21 where the GalNAc ligand is conjugated to the 3′/5′-terminal of theadditional 3′ and/or 5′ nucleotide spacers. Guide RNA strand is extendedto 3′-end or to the 5′-end or both ends with desired number ofnucleotides. (GalNAc) is conjugated to the 3′-end, 5′-end or both endsof oligonucleotide that is (are) complementary to the extendednucleotides on the guide RNA strand to form complementary duplex leadingto a single chemical entity. The conjugate designs 3-3 to 3-8 areconstructed from extended nucleotide spacers and the spacercomplementary strand carrying a GalNAc ligand. As used in Table 3,uppercase A, C, G and U refer to ribonucleotides bearing nucleobasesadenine, cytosine, guanidine and uracil, respectively; lowercase a, c, gand u refer to modified (e.g., 2′-OMe or 2′-MOE) ribonucleotides bearingnucleobases adenine, cytosine, guanidine and uracil, respectively;letter “T” refers to thymidine or deoxythymidine; and letter “s” refersto a phosphate linkage (such as a phosphorothioate (PS) linkage, aphosphodiester linkage, or a phosphorodithioate linkage). In someembodiments, “s” represents a PS linkages. As used in Table 3,“(GalNAc)” refers to a targeting moiety such as one comprising a GalNAcor a derivative thereof. As used in Table 3, “(GalNAc)” also encompassesa targeting moiety that comprises multiple GalNAc structures orderivatives thereof such as a dimer, trimer, a tetramer of GalNAc orderivatives thereof.

TABLE 3 Guide RNA GalNAc conjugate designs Conjugate SEQ RNA GalNAc No.ID No conjugate designs 3-1 675′-gsgscsUGAUGAG GCCGCACAUG GUUUUAGAgc uagaaauagcAAGUUAAAAU AAGGCUAGUC CGUUAUCAac uugaaaaaguggcaccgagu cggugcuususus-(GalNAc3)-3′ 3-2 685′-gsgscsUsGAUsGAG GCCGCsACsAUG GUUUUsAGAgcusagaaausagc AAGUUsAAAAUs AAGGCUsAGUC CGUUsAUCsAacuusgaaaaagus ggcaccgagu cggugcuuusus-(GalNAc3)-3′ 3-3′ 695′-gsgscsUGAUGAG GCCGCACAUG GUUUUAGAgc uagaaauagcAAGUUAAAAU AAGGCUAGUC CGUUAUCAac uugaaaaaguggcaccgagu cggugcusususuuuccuuuguuuuugsc3′ 71 3′gsgsaaacaaaaacgs(GalNAc)3-4 70 5′-cscuuuguuuuugcuugsgscsUGAUGAG GCCGCACAUGGUUUUAGAgc uagaaauagc AAGUUAAAAU AAGGCUAGUCCGUUAUCAac uugaaaaagu ggcaccgagu cggugcusususu3′gsgsaaacaaaaacgs(GalNAc) 3′gsgsaaacaaaaacgs(GalNAc) (SEQ ID NO 71)(SEQ ID NO 72) 3-5 73 5′-cscuuuguuuuugcuugsgscsUGAUGAGGCCGCACAUGGUUUUAGAgc uagaaauagc AAGUUAAAAU AAGGCUAGUCCGUUAUCAac uugaaaaagu ggcaccgagu cggugcusususuuuccuuuguuuuugsc-3′3′gsgsaaacaaaaacgs(GalNAc) 3′gsgsaaacaaaaacgs(GalNAc) (SEQ ID NO 74)(SEQ ID NO 75) 3-6 76 5′-gsgscsUGAUGAG GCCGCACAUG GUUUUAGAgc uagaaauagcAAGUUAAAAU AAGGCUAGUC CGUUAUCAac uugaaaaaguggcaccgagu cggugcusususuTTTccuuuguuuuugsc3′ 773′gsgsaaacaaaaacgs(GalNAc) 3-7 785′-cscuuuguuuuugcTTTgsgscsUGAUGAG GCCGCACAUGGUUUUAGAgc uagaaauagc AAGUUAAAAU AAGGCUAGUCCGUUAUCAac uugaaaaagu ggcaccgagu cggugcusususu 793′gsgsaaacaaaaacgs(GalNAc) 3-8 805′-cscuuuguuuuugcTTTgsgscsUGAUGAG GCCGCACAUGGUUUUAGAgc uagaaauagc AAGUUAAAAU AAGGCUAGUCCGUUAUCAac uugaaaaagu ggcaccgagu cggugcusususuTTTccuuuguuuuugsc-3′3′gsgsaaacaaaaacgs(GalNAc) 3′gsgsaaacaaaaacgs(GalNAc) (SEQ ID NO 81)(SEQ ID NO 82) 3-9 83 5′-(GalNAc)uuugsgscsUGAUGAG GCCGCACAUG GUUUUAGAgcuagaaauagc AAGUUAAAAU AAGGCUAGUC CGUUAUCAacuugaaaaagu ggcaccgagu cggugcuususus-(GalNAc3)-3′ 3-10 845′-(GalNAc)uuugsgscsUsGAUsGAG GCCGCsACsAUGGUUUUsAGAgc usagaaausagc AAGUUsAAAAUs AAGGCUsAGUCCGUUsAUCsAac uusgaaaaagus ggcaccgagu cggugcuuusus- (GalNAc3)-3′ 3-11 855′-(GalNAc)uuugsgscsUGAUGAG GCCGCACAUG GUUUUAGAgcuagaaauagc AAGUUAAAAU AAGGCUAGUC CGUUAUCAacuugaaaaagu ggcaccgagu cggugcusususu-3′ 3-12 865′-(GalNAc)uuugsgscsUsGAUsGAG GCCGCsACsAUGGUUUUsAGAgc usagaaausagc AAGUUsAAAAUs AAGGCUsAGUCCGUUsAUCsAac uusgaaaaagus ggcaccgagu cggugcuuusus-3′ 3-13 875′-(GalNAc)TTTgsgscsUGAUGAG GCCGCACAUG GUUUUAGAgcuagaaauagc AAGUUAAAAU AAGGCUAGUC CGUUAUCAacuugaaaaagu ggcaccgagu cggugcuususus-(GalNAc3)-3 3-14 885′-(GalNAc)TTTgsgscsUsGAUsGAG GCCGCsACsAUGGUUUUsAGAgc usagaaausagc AAGUUsAAAAUs AAGGCUsAGUCCGUUsAUCsAac uusgaaaaagus ggcaccgagu cggugcuuusus-(GalNAc3)-3′ 3-15 895′-(GalNAc)TTTgsgscsUGAUGAG GCCGCACAUG GUUUUAGAgcuagaaauagc AAGUUAAAAU AAGGCUAGUC CGUUAUCAacuugaaaaagu ggcaccgagu cggugcusususu-3′ 3-16 905′-(GalNAc)TTTgsgscsUsGAUsGAG GCCGCsACsAUGGUUUUsAGAgc usagaaausagc AAGUUsAAAAUs AAGGCUsAGUCCGUUsAUCsAac uusgaaaaagus ggcaccgagu cggugcuuusus-3′ 3-17 915′-gsgscsUGAUGAG GCCGCACAUG GUUUUAGAgc uagaaauagcAAGUUAAAAU AAGGCUAGUC CGUUAUCAac uugaaaaaguggcaccgagu cggugcuusususTTTs(GalNAc3)-3′ 3-18 925′-gsgscsUsGAUsGAG GCCGCsACsAUG GUUUUsAGAgcusagaaausagc AAGUUsAAAAUs AAGGCUsAGUC CGUUsAUCsAacuusgaaaaagus ggcaccgagu cggugcuuususTTTs- (GalNAc3)-3′ 3-19 935′-gsgscsUGAUGAG GCCGCACAUG GUUUUAGAgc uagaaauagcAAGUUAAAAU AAGGCUAGUC CGUUAUCAac uugaaaaaguggcaccgagu cggugcuususussususuUUUs(GalNAc3)-3′ 3-20 945′-gsgscsUsGAUsGAG GCCGCsACsAUG GUUUUsAGAgcusagaaausagc AAGUUsAAAAUs AAGGCUsAGUC CGUUsAUCsAacuusgaaaaagus ggcaccgagu cggugcuuusussususuUUUs- (GalNAc3)-3′ 3-21 955′-gsgscsUGAUGAG GCCGCACAUG GUUUUAGAgc uagaaauagcAAGUUAAAAU AAGGCUAGUC CGUUAUCAac uugaaaaaguggcaccgagu cggugcuususussususuuuus(GalNAc3)-3′ 3-22 965′-aaaaaaaaaaaaaaaaaTTTgsgscsUGAUGAG GCCGCACAUGGUUUUAGAgc uagaaauagc AAGUUAAAAU AAGGCUAGUCCGUUAUCAac uugaaaaagu ggcaccgagu cggugcusususuTTTaaaaaaaaaaaaaaaaa-3′ 973′- 3′-ususuuuuuuuu ususuuuuuuuuu uuuuuuus(GalNAc)-5′ uuuuuus(GalNAc)-5′3-23 98 5′-gsgscsUGAUGAG GCCGCACAUG GUUUUAGAgc uagaaauagcAAGUUAAAAU AAGGCUAGUC CGUUAUCAac uugaaaaaguggcaccgagu cggugcusususuaaaaaaaaaaaaaaaaa-3′ 993′-ususuuuuuuuuuuuuuuus(GalNAc)-5′ 3-24 1005′-gsgscsUGAUGAG GCCGCACAUG GUUUUAGAgc uagaaauagcAAGUUAAAAU AAGGCUAGUC CGUUAUCAac uugaaaaaguggcaccgagu cggugcusususuAAAAAAAAAAAAAAAAAAAA-3′ 1013′-ususuuuuuuuuuuuuuuus(GalNAc)-5′ 3-25 1025′-gsgscsUGAUGAG GCCGCACAUG GUUUUAGAgc uagaaauagcAAGUUAAAAU AAGGCUAGUC CGUUAUCAac uugaaaaaguggcaccgagu cggugcusususuAAAAAAAAAAAAAAAAAAAA-3′ 1033′-ususuuuuuuuuuuuuuuuuuus(GalNAc)-5′

As disclosed herein, the nucleotide sequences and modification patternsencompass all length, structure, and type of RNAs or fragments thereof,CRISPR guide RNAs, e.g. sgRNAs, dual guide RNAs, or mRNAs. For example,nucleotide sequences and modification patterns as described in Table 3above may indicate RNA sequences and modification patterns in a singleguide RNA, a dual guide RNA, a nuclease mRNA, or any fragment or segmentthereof.

A targeting moiety can be attached to a nucleic acid described hereinvia a carrier. The carriers may include (i) at least one “backboneattachment point,” preferably two “backbone attachment points” and (ii)at least one “tethering attachment point.” A “backbone attachment point”as used herein refers to a functional group, e.g. a hydroxyl group, orgenerally, a bond available for, and that is suitable for incorporationof the carrier monomer into the backbone, e.g., the phosphate, ormodified phosphate, e.g., sulfur containing, backbone, of anoligonucleotide. A “tethering attachment point” (TAP) in refers to anatom of the carrier monomer, e.g., a carbon atom or a heteroatom(distinct from an atom which provides a backbone attachment point), thatconnects a selected moiety. The selected moiety can be, e.g., acarbohydrate, e.g. monosaccharide, disaccharide, trisaccharide,tetrasaccharide, oligosaccharide and polysaccharide. Optionally, theselected moiety is connected by an intervening tether to the carriermonomer. Thus, the carrier will often include a functional group, e.g.,an amino group, or generally, provide a bond, that is suitable forincorporation or tethering of another chemical entity, e.g., a ligand tothe constituent atom. Representative U.S. patents that teach thepreparation of conjugates of nucleic acids include, but are not limitedto, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465;5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731;5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603;5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025;4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582;4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963;5,149,782; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;5,599,928; 5,672,662; 5,688,941; 5,714,166; 6,153,737; 6,172,208;6,300,319; 6,335,434; 6,335,437; 6,395,437; 6,444,806; 6,486,308;6,525,031; 6,528,631; 6,559,279; contents of which are hereinincorporated in their entireties by reference.

A targeting moiety can be attached to an active agent, e. g. a guideRNA, via a linker. A linker may be bound to one or more active agentsand a targeting moiety ligand to form a conjugate, wherein the conjugatereleases at least one active agent, e.g. a guide RNA or guide RNA-Cascomplex, upon delivery to a target cell. The linker may be attached tothe targeting moiety and the active agent by functional groupsindependently selected from an ester bond, disulfide, amide,acylhydrazone, ether, carbamate, carbonate, and urea. Alternatively thelinker can be attached to either the targeting moiety or the activeagent by a non-cleavable group such as provided by the conjugationbetween a thiol and a maleimide, an azide and an alkyne. In someembodiments, a targeting moiety comprises one or more linkers. In someembodiments, one or more linkers as described herein connect a portionof the targeting moiety to a different portion of the targeting moiety.For example, a targeting moiety can comprise 2, 3, 4, 5 or more GalNAcstructures or derivatives thereof that are connected by one or morelinkers. In some embodiments, two or more GalNAc structures orderivatives thereof in a targeting moiety are connected by one or morenon-cleavable linkers. In some embodiments, a herein described conjugatecomprises an active agent that is directly connected to a sugar moietyof the targeting moiety.

The linkers can each independently comprises one or more functionalgroups selected from the group consisting of ethylene glycol, propyleneglycol, amide, ester, ether, alkyl, cycloalkyl, heterocyclyl, aryl, andheteroaryl, wherein each of the alkyl, alkenyl, cycloalkyl,heterocyclyl, aryl, and heteroaryl groups optionally is substituted withone or more groups, each independently selected from halogen, cyano,nitro, hydroxyl, carboxyl, carbamoyl, ether, alkoxy, aryloxy, amino,amide, carbamate, alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl,heteroaryl, heterocyclyl, wherein each of the carboxyl, carbamoyl,ether, alkoxy, aryloxy, amino, amide, carbamate, alkyl, alkenyl,alkynyl, aryl, arylalkyl, cycloalkyl, heteroaryl, or heterocyclyl isoptionally substituted with one or more groups, each independentlyselected from halogen, cyano, nitro, hydroxyl, carboxyl, carbamoyl,ether, alkoxy, aryloxy, amino, amide, carbamate, alkyl, alkenyl,alkynyl, aryl, arylalkyl, cycloalkyl, heteroaryl, heterocyclyl. In someembodiments, a linker independently comprises phosphate,phosphorothioate, amide, ether, oxime, hydrazine or carbamate. Ascontemplated herein it should be understood that, in some embodiments, atargeting conjugate of Formula (V), (VI), (VIa) or (VIb) comprises alinker described herein. For example, any of the groups R and L¹-L¹² cancomprise one or more linkers.

In some embodiments, the linker can independently comprise a C₁-C₁₀straight chain alkyl, C₁-C₁₀ straight chain O-alkyl, C₁-C₁₀ straightchain substituted alkyl, C₁-C₁₀ straight chain substituted O-alkyl,C₄-C₁₃ branched chain alkyl, C₄-C₁₃ branched chain O-alkyl, C₂-C₁₂straight chain alkenyl, C₂-C₁₂ straight chain O-alkenyl, aralkyl, C₃-C₁₂straight chain substituted alkenyl, C₃-C₁₂ straight chain substitutedO-alkenyl, polyethylene glycol, polylactic acid, polygly colic acid,poly(lactide-co-glycolide), polycarprolactone, polycyanoacrylate,ketone, aryl, heterocyclic, succinic ester, amino acid, aromatic group,ether, crown ether, urea, thiourea, amide, purine, pyrimidine,bypiridine, indole derivative acting as a cross linker, chelator,aldehyde, ketone, bisamine, bis alcohol, heterocyclic ring structure,azirine, disulfide, thioether, hydrazone and combinations thereof. Forexample, the linker can be a C3 straight chain alkyl or a ketone. Thealkyl chain of the linker can be substituted with one or moresubstituents or heteroatoms. In some embodiments, the alkyl chain of thelinker may optionally be interrupted by one or more atoms or groupsselected from —O—, —C(═O)—, —NR, —O—C(═O)—NR—, —S—, —S—S—.

In some embodiments, the linker may be cleavable and is cleaved torelease the active agent. The cleavable functionality may be hydrolyzedin vivo or may be designed to be hydrolyzed enzymatically, for exampleby Cathepsin B. A “cleavable” linker, as used herein, refers to anylinker which can be cleaved physically or chemically. Examples forphysical cleavage may be cleavage by light, radioactive emission orheat, while examples for chemical cleavage include cleavage byre-dox-reactions, hydrolysis, pH-dependent cleavage.

Linkers may comprise a direct bond or an atom such as oxygen or sulfur,a unit such as NR¹, C(O), C(O)NH, SO, SO2, SO2NH or a chain of atoms,such as, but not limited to, substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl,heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl,heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl,heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl,alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl,alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl,alkenylheteroarylalkenyl, alkenylheteroarylalkynyl,alkynylheteroarylalkyl, alkynylheteroarylalkenyl,alkynylheteroarylalkynyl, alkylheterocyclylalkyl,alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl,alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl,alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl,alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl,alkynylhereroaryl, which one or more methylenes can be interrupted orterminated by O, S, S(O), SO2, N(R′), C(O), substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted heterocyclic; where R′ is hydrogen, acyl, aliphatic orsubstituted aliphatic. In one embodiment, the linker is between 1-24atoms, preferably 4-24 atoms, preferably 6-18 atoms, more preferably8-18 atoms, and most preferably 8-16 atoms.

In one embodiment, the linker is —[(P-Q″-R)q-X—(P′Q′″-R′)q′]q″-T-,wherein P, R, T, P′, R′ and T are each independently for each occurrenceabsent, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH, CH₂O; NHCH(Ra)C(O),—C(O)—CH(Ra)—NH—, CH═N—O,

or heterocyclyl; Q″ and Q′″ are each independently for each occurrenceabsent, —(CH2)n-, —C(R1)(R2)(CH2)n-, —(CH2)nC(R1)(R2)-,(CH2CH2O)mCH2CH2-, or —(CH2CH2O)mCH2CH2NH—; X is absent or a cleavablelinking group; Ra is H or an amino acid side chain; R1 and R2 are eachindependently for each occurrence H, CH3, OH, SH or N(RN)2; RN isindependently for each occurrence H, methyl, ethyl, propyl, isopropyl,butyl or benzyl; q, q′ and q″ are each independently for each occurrence0-20 and wherein the repeating unit can be the same or different; n isindependently for each occurrence 1-20; and m is independently for eachoccurrence 0-50.

In one embodiment, the linker comprises at least one cleavable linkinggroup. In certain embodiments, the linker is a branched linker. Thebranchpoint of the branched linker may be at least trivalent, but may bea tetravalent, pentavalent or hexavalent atom, or a group presentingsuch multiple valencies. In certain embodiments, the branchpoint is, —N,—N(O)—C, —O—C, —S—C, —SS—C, —C(O)N(O)—C, —OC(O)N(O)—C, —N(O)C(O)—C, orN(O)C(O)O—C; wherein Q is independently for each occurrence H oroptionally substituted alkyl. In other embodiment, the branchpoint isglycerol or glycerol derivative.

In one embodiment, a linker may be cleaved by an enzyme. As anon-limiting example, the linker may be a polypeptide moiety, e.g. AA inWO2010093395 to Govindan, the content of which is incorporated herein byreference in its entirety; that is cleavable by intracellular peptidase.Govindan teaches AA in the linker may be a di, tri, or tetrapeptide suchas Ala-Leu, Leu-Ala-Leu, and Ala-Leu-Ala-Leu. In another example, thecleavable linker may be a branched peptide. The branched peptide linkermay comprise two or more amino acid moieties that provide an enzymecleavage site. Any branched peptide linker disclosed in WO 1998019705 toDubowchik, the content of which is incorporated herein by reference inits entirety, may be used as a linker in the conjugate of the presentdisclosure. As another example, the linker may comprise a lysosomallycleavable polypeptide disclosed in U.S. Pat. No. 8,877,901 to Govindanet al., the content of which is incorporated herein by reference in itsentirety. As another example, the linker may comprise a protein peptidesequence which is selectively enzymatically cleavable by tumorassociated proteases, such as any Y and Z structures disclosed in U.S.Pat. No. 6,214,345 to Firestone et al, the content of which isincorporated herein by reference in its entirety.

In some embodiments, a linker may comprise a cleavable linking group. Acleavable linking group is one which is sufficiently stable outside thecell, but which upon entry into a target cell is cleaved to release thetwo parts the linker is holding together. In a preferred embodiment, thecleavable linking group is cleaved at least 10 times or more, preferablyat least 100 times faster in the target cell or under a first referencecondition (which can, e.g., be selected to mimic or representintracellular conditions) than in the blood of a subject, or under asecond reference condition (which can, e.g., be selected to mimic orrepresent conditions found in the blood or serum). Cleavable linkinggroups may be susceptible to cleavage agents, e.g., pH, redox potentialor the presence of degradative molecules. Generally, cleavage agents aremore prevalent or found at higher levels or activities inside cells thanin serum or blood. Examples of such degradative agents include: redoxagents which are selected for particular substrates or which have nosubstrate specificity, including, e.g., oxidative or reductive enzymesor reductive agents such as mercaptans, present in cells, that candegrade a redox cleavable linking group by reduction; esterases;endosomes or agents that can create an acidic environment, e.g., thosethat result in a pH of five or lower; enzymes that can hydrolyze ordegrade an acid cleavable linking group by acting as a general acid,peptidases (which can be substrate specific), and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptibleto pH. The pH of human serum is 7.4, while the average intracellular pHis slightly lower, ranging from about 7.1-7.3. Endosomes have a moreacidic pH, in the range of 5.5-6.0, and lysosomes have an even moreacidic pH at around 5.0. Some linkers will have a cleavable linkinggroup that is cleaved at a preferred pH, thereby releasing the cationiclipid from the ligand inside the cell, or into the desired compartmentof the cell.

A linker can include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the cell to be targeted. For example, livertargeting ligands can be linked to the cationic lipids through a linkerthat includes an ester group. Liver cells are rich in esterases, andtherefore the linker will be cleaved more efficiently in liver cellsthan in cell types that are not esterase-rich. Other cell-types rich inesterases include cells of the lung, renal cortex, and testis.

One class of cleavable linking groups are redox cleavable linking groupsthat are cleaved upon reduction or oxidation. An example of reductivelycleavable linking group is a disulphide linking group (—S—S—). Todetermine if a candidate cleavable linking group is a suitable“reductively cleavable linking group,” or for example is suitable foruse with a particular RNA moiety and particular targeting agent one canlook to methods described herein. For example, a candidate can beevaluated by incubation with dithiothreitol (DTT), or other reducingagent using reagents know in the art, which mimic the rate of cleavagewhich would be observed in a cell, e.g., a target cell. The candidatescan also be evaluated under conditions which are selected to mimic bloodor serum conditions. In a preferred embodiment, candidate compounds arecleaved by at most 10% in the blood. In preferred embodiments, usefulcandidate compounds are degraded at least 2, 4, 10 or 100 times fasterin the cell (or under in vitro conditions selected to mimicintracellular conditions) as compared to blood (or under in vitroconditions selected to mimic extracellular conditions). The rate ofcleavage of candidate compounds can be determined using standard enzymekinetics assays under conditions chosen to mimic intracellular media andcompared to conditions chosen to mimic extracellular media.

In some embodiments, a linker may comprise a phosphate based cleavablelinking group. Phosphate-based cleavable linking groups are cleaved byagents that degrade or hydrolyze the phosphate group. An example of anagent that cleaves phosphate groups in cells are enzymes such asphosphatases in cells. Examples of phosphate-based linking groups (i.e.,phosphorus-containing linkages or phosphorus-containing linkers) are—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—,—O—P(O)(ORk)-S—, S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—,—O—P(O)(Rk)-O—, O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—,—S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—. In some embodiments, phosphate-basedlinking groups are —OP(O)(OH) O—, —O—P(S)(OH)-O—, —O—P(S)(SH)-O—,—S—P(O)(OH)-O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—,—S—P(S)(OH)-O—, —O—P(O)(H)-O—, —O—P(S)(H)-O—, —S—P(O)(H)-O—,—S—P(S)(H)-O—, —S—P(O)(H)—S—, —O—P(S)(H)—S—S—. In some embodiments, aphosphate-based linker is O—P(O)(OH)-O—.

In some embodiments, a linker may comprise an acid cleavable linkinggroup. Acid cleavable linking groups are linking groups that are cleavedunder acidic conditions. In preferred embodiments acid cleavable linkinggroups are cleaved in an acidic environment with a pH of about 6.5 orlower (e.g., about 6.0, 5.5, 5.0, or lower), or by agents such asenzymes that can act as a general acid. In a cell, specific low pHorganelles, such as endosomes and lysosomes can provide a cleavingenvironment for acid cleavable linking groups. Examples of acidcleavable linking groups include but are not limited to hydrazones,esters, and esters of amino acids. Acid cleavable groups can have thegeneral formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is whenthe carbon attached to the oxygen of the ester (the alkoxy group) is anaryl group, substituted alkyl group, or tertiary alkyl group such asdimethyl pentyl or t-butyl. These candidates can be evaluated usingmethods analogous to those described above.

In some embodiments, a linker may comprise a ester based linking group.Ester-based cleavable linking groups are cleaved by enzymes such asesterases and amidases in cells. Examples of ester-based cleavablelinking groups include but are not limited to esters of alkylene,alkenylene and alkynylene groups. Ester cleavable linking groups havethe general formula —C(O)O—, or —OC(O)—. These candidates can beevaluated using methods analogous to those described above.

In some embodiments, a linker may comprise a peptide based linkinggroup. Peptide-based cleavable linking groups are cleaved by enzymessuch as peptidases and proteases in cells. Peptide-based cleavablelinking groups are peptide bonds formed between amino acids to yieldoligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides.Peptide-based cleavable groups do not include the amide group(—C(O)NH—). The amide group can be formed between any alkylene,alkenylene or alkynelene. A peptide bond is a special type of amide bondformed between amino acids to yield peptides and proteins. The peptidebased cleavage group is generally limited to the peptide bond (i.e., theamide bond) formed between amino acids yielding peptides and proteinsand does not include the entire amide functional group. Peptide-basedcleavable linking groups have the general formula—NHCHRAC(O)NHCHRBC(O)—, where RA and RB are the R groups of the twoadjacent amino acids.

Linkers that contain peptide bonds can be used when targeting cell typesrich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group canbe evaluated by testing the ability of a degradative agent (orcondition) to cleave the candidate linking group. It will also bedesirable to also test the candidate cleavable linking group for theability to resist cleavage in the blood or when in contact with othernon-target tissue. Thus one can determine the relative susceptibility tocleavage between a first and a second condition, where the first isselected to be indicative of cleavage in a target cell and the second isselected to be indicative of cleavage in other tissues or biologicalfluids, e.g., blood or serum. The evaluations can be carried out in cellfree systems, in cells, in cell culture, in organ or tissue culture, orin whole animals. It may be useful to make initial evaluations incell-free or culture conditions and to confirm by further evaluations inwhole animals. In preferred embodiments, useful candidate compounds arecleaved at least 2, 4, 10 or 100 times faster in the cell (or under invitro conditions selected to mimic intracellular conditions) as comparedto blood or serum (or under in vitro conditions selected to mimicextracellular conditions).

In some embodiments, a herein described conjugate comprises a structureof Formula (I),

wherein each X is independently H or a protecting group, and Wrepresents an active agent or a coupling sequence. The one or morelinkers of Formula (I) can each independently comprises a linker asdescribed in this disclosure. In some embodiments, each of theprotecting group of Formula (I) is independently selected from:4-acetoxy-2,2-dimethylbutanoyl (ADMB),3-(2-Hydroxyphenyl)-3,3-dimethylpropanoate (DMBPP),3-(2-hydroxy-4,6-dimethylphenyl)-3,3-dimethylpropanoate groups (TMBPP),methyl sulfonylethoxycarbonyl (Msc), 2,2-dimethyltrimethylene (DMTM)phosphate, 2-pyridylmethyl, ethyl mandelate, (phenylthiomethyl)benzyl,pentafluoropropionyl (PFP), benzoyl (Bz), acetyl (Ac), bacillosamine(Bac), benzyl (Bn), 1-b enzenesulfinylpiperidine (BSP),tert-butoxycarbonyl (Boc), benzylidene acetal, propargyl,naphthylpropargyl, carbonate, dichloroacetyl, tert-butylsilylene,tetraisopropyldisiloxanylidene (TIPDS), methoxybenzyl (PMB), xylylene,and p-methoxyphenyl (MP). Exemplary protecting groups are furtherdisclosed in Guo et al., Molecules 2010, 15, 7235-7265, which is herebyincorporated by reference in its entirety. In some embodiments, X is H.In some embodiments, each X is independently selected from H and Bz. Insome embodiments of Formula (I), W is an active agent. In someembodiments, W is a nucleic acid. In some embodiments, W is a gRNA. Insome embodiments, W is a single-stranded, double-stranded, partiallydouble-stranded or hairpin stem-loop nucleic acid. In some embodimentsof Formula (I), W is a coupling sequence. In some embodiments, Wcomprises an RNA or DNA sequence. W can comprise one or more modifiedDNA or RNA bases. The nucleobases can comprise any chemicalmodifications as described herein. In some embodiments, the nucleobasesinclude a 2′-OH or 2′-OMe modification. For example, W may comprise oneor more 2′-OMe modified adenine, cytosine, guanidine and uracil,referred to as (a) (c), (g), or (u). In some embodiments, a modifiedRNA, e.g. a gRNA or mRNA, includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50 or more modifiednucleobases. In some embodiments, a modified RNA comprises one or moremodified nucleobases near the 5′ end, near the 3′ end, or in the middleof the sequence. The modified nucleobases within a modified RNA may ormay not be contiguous. In some embodiments, a modified RNA comprises oneor more 2′-OMe modifications scattered along the length of the sequence.In some embodiments, a modified RNA comprises one or more 2′OHmodifications scattered along the length of the sequence. In someembodiments, a modified RNA comprises alternating 2′-OH and 2′OMemodifications. In some embodiments, W comprises (A)n, (T)n, (U)n, (a)n,or (u)n, wherein n is an integer no less than 3, wherein a is2′-O-methyladenosine (2′-OMe A), and wherein u is 2′-O-methyluridine(2′-OMe-U). In some embodiments, W comprises (u)n, wherein n is aninteger from 3 to 50 (SEQ ID NO: 118). In some embodiments, W comprises(u)n, wherein n is an integer from 3 to 20 (SEQ ID NO: 119) or 3 to 15(SEQ ID NO: 120). In some embodiments, W comprises one or morenucleotide sequences that are complementary to a coupling sequence. Insome embodiments, W comprises one or more guanines or cytidines. In someembodiments, W comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 20, 25, 30, 35, 40, 50 or more guanines or cytidines. Insome embodiments, the one or more guanines or cytidines arecomplementary to one or more cytidines or guanines in a couplingsequence. In some embodiments, the guanines or cytidines are at theterminals of W or the coupling sequence. Not intended to be bound by anytheory, it is contemplated that the guanines-cytidine pairing forms “GClocks” or “CG locks” that would increase binding affinity. The guaninesand/or cytidines in W or a coupling sequence may or may not becontiguous and may comprise any one of the chemical modifications asdescribed herein, e.g. a 2′-OMe or 2′-OH modification.

In some embodiments, a conjugate of Formula (I) comprises a structure ofFormula (Ia),

In some embodiments, a conjugate of Formula (I) comprises a structure ofFormula (Ib),

In some embodiments, a herein described conjugate comprises a structureof Formula (II),

wherein each X is independently H or a protecting group, Z is modifiedor unmodified C₅ or C₆ monosaccharide, and W represents an active agentor a coupling sequence. The one or more linkers of Formula (II) can eachindependently comprises a linker as described in this disclosure. Insome embodiments, each of the protecting group of Formula (II) isindependently selected from: 4-acetoxy-2,2-dimethylbutanoyl (ADMB),3-(2-Hydroxyphenyl)-3,3-dimethylpropanoate (DMBPP),3-(2-hydroxy-4,6-dimethylphenyl)-3,3-dimethylpropanoate groups (TMBPP),methyl sulfonylethoxycarbonyl (Msc), 2,2-dimethyltrimethylene (DMTM)phosphate, 2-pyridylmethyl, ethyl mandelate, (phenylthiomethyl)benzyl,pentafluoropropionyl (PFP), benzoyl (Bz), acetyl (Ac), bacillosamine(Bac), benzyl (Bn), 1-benzenesulfinylpiperidine (BSP),tert-butoxycarbonyl (Boc), benzylidene acetal, propargyl,naphthylpropargyl, carbonate, dichloroacetyl, tert-butylsilylene,tetraisopropyldisiloxanylidene (TIPDS), methoxybenzyl (PMB), xylylene,and p-methoxyphenyl (MP). In some embodiments, X is H. In someembodiments, each X is independently selected from H and Bz. In someembodiments of Formula (II), Z is galactose or mannose. In someembodiments of Formula (II), Z is GalNAc. In some embodiments of Formula(II), W is an active agent. In some embodiments, W is a nucleic acid. Insome embodiments, W is a gRNA. In some embodiments, W is asingle-stranded, double-stranded, partially double-stranded or hairpinstem-loop nucleic acid. In some embodiments of Formula (II), W is acoupling sequence. In some embodiments, W comprises an RNA or DNAsequence. In some embodiments, W comprises (A)n, (T)n, (U)n, (a)n, or(u)n, wherein n is an integer no less than 3, wherein a is2′-O-methyladenosine (2′-OMe A), and wherein u is 2′-O-methyluridine(2′-OMe-U). In some embodiments, W comprises (u)n, wherein n is aninteger from 3 to 50 (SEQ ID NO: 118). In some embodiments, W comprises(u)n, wherein n is an integer from 3 to 20 (SEQ ID NO: 119) or 3 to 15(SEQ ID NO: 120).

In some embodiments, a conjugate of Formula (II) comprises a structureof Formula (IIa),

In some embodiments, a conjugate of Formula (II) comprises a structureof Formula (IIb),

In some embodiments, a conjugate of Formula (II) comprises a structureof Formula (IIc),

In some embodiments, a herein described conjugate comprises a structureof Formula (III),

wherein each X is independently H or a protecting group, Z is modifiedor unmodified C₅ or C₆ monosaccharide, and W represents an active agentor a coupling sequence. The one or more linkers of Formula (III) caneach independently comprises a linker as described in this disclosure.In some embodiments, each of the protecting group of Formula (III) isindependently selected from: 4-acetoxy-2,2-dimethylbutanoyl (ADMB),3-(2-Hydroxyphenyl)-3,3-dimethylpropanoate (DMBPP),3-(2-hydroxy-4,6-dimethylphenyl)-3,3-dimethylpropanoate groups (TMBPP),methyl sulfonylethoxycarbonyl (Msc), 2,2-dimethyltrimethylene (DMTM)phosphate, 2-pyridylmethyl, ethyl mandelate, (phenylthiomethyl)benzyl,pentafluoropropionyl (PFP), benzoyl (Bz), acetyl (Ac), bacillosamine(Bac), benzyl (Bn), 1-benzenesulfinylpiperidine (BSP),tert-butoxycarbonyl (Boc), benzylidene acetal, propargyl,naphthylpropargyl, carbonate, dichloroacetyl, tert-butylsilylene,tetraisopropyldisiloxanylidene (TIPDS), methoxybenzyl (PMB), xylylene,and p-methoxyphenyl (MP). In some embodiments, X is H. In someembodiments, each X is independently selected from H and Bz. In someembodiments of Formula (III), Z is galactose or mannose. In someembodiments of Formula (III), Z is GalNAc. In some embodiments ofFormula (III), W is an active agent. In some embodiments, W is a nucleicacid. In some embodiments, W is a gRNA. In some embodiments, W is asingle-stranded, double-stranded, partially double-stranded or hairpinstem-loop nucleic acid. In some embodiments of Formula (III), W is acoupling sequence. In some embodiments, W comprises an RNA or DNAsequence. In some embodiments, W comprises (A)n, (T)n, (U)n, (a)n, or(u)n, wherein n is an integer no less than 3, wherein a is2′-O-methyladenosine (2′-OMe A), and wherein u is 2′-O-methyluridine(2′-OMe-U). In some embodiments, W comprises (u)n, wherein n is aninteger from 3 to 50 (SEQ ID NO: 118). In some embodiments, W comprises(u)n, wherein n is an integer from 3 to 20 (SEQ ID NO: 119) or 3 to 15(SEQ ID NO: 120).

In some embodiments, a conjugate of Formula (III) comprises a structureof Formula (IIIa),

In some embodiments, a conjugate of Formula (III) comprises a structureof Formula (IIIb),

In some embodiments, a conjugate of Formula (III) comprises a structureof Formula (IIC),

wherein Y is O or S.

In some embodiments, a conjugate of Formula (III) comprises a structureof Formula (IIId),

wherein Y is O or S.

In some embodiments, a conjugate of Formula (III) comprises a structureof Formula (IIIe),

wherein Y is O or S.

In some embodiments, a herein described conjugate comprises a structureof Formula

wherein each X is independently H or a protecting group, R^(A) is —OX or—NHAc, Y is O or S, and W represents an active agent or a couplingsequence. The one or more linkers of Formula (IV) can each independentlycomprises a linker as described in this disclosure. In some embodiments,each of the protecting group of Formula (IV) is independently selectedfrom: 4-acetoxy-2,2-dimethylbutanoyl (ADMB),3-(2-Hydroxyphenyl)-3,3-dimethylpropanoate (DMBPP),3-(2-hydroxy-4,6-dimethylphenyl)-3,3-dimethylpropanoate groups (TMBPP),methyl sulfonylethoxycarbonyl (Msc), 2,2-dimethyltrimethylene (DMTM)phosphate, 2-pyridylmethyl, ethyl mandelate, (phenylthiomethyl)benzyl,pentafluoropropionyl (PFP), benzoyl (Bz), acetyl (Ac), bacillosamine(Bac), benzyl (Bn), 1-benzenesulfinylpiperidine (BSP),tert-butoxycarbonyl (Boc), benzylidene acetal, propargyl,naphthylpropargyl, carbonate, dichloroacetyl, tert-butylsilylene,tetraisopropyldisiloxanylidene (TIPDS), methoxybenzyl (PMB), xylylene,and p-methoxyphenyl (MP). In some embodiments, X is H. In someembodiments, each X is independently selected from H and Bz. In someembodiments, R^(A) is —OX. In some embodiments, R^(A) is —OH. In someembodiments, R^(A) is —NHAc. In some embodiments of Formula (IV), W isan active agent. In some embodiments, W is a nucleic acid. In someembodiments, W is a gRNA. In some embodiments, W is a single-stranded,double-stranded, partially double-stranded or hairpin stem-loop nucleicacid. In some embodiments of Formula (IV), W is a coupling sequence. Insome embodiments, W comprises an RNA or DNA sequence. In someembodiments, W comprises (A)n, (T)n, (U)n, (a)n, or (u)n, wherein n isan integer no less than 3, wherein a is 2′-O-methyladenosine (2′-OMe A),and wherein u is 2′-O-methyluridine (2′-OMe-U). In some embodiments, Wcomprises (u)n, wherein n is an integer from 3 to 50 (SEQ ID NO: 118).In some embodiments, W comprises (u)n, wherein n is an integer from 3 to20 (SEQ ID NO: 119) or 3 to 15 (SEQ ID NO: 120).

In some embodiments, a conjugate of Formula (IV) comprises a structureof 1-1, 1-2, 1-5, 1-6, 1-9, 1-10, 1-11, or 1-12 as shown in Table 1.

In some embodiments of Formula (I), Formula (Ia), Formula (II), Formula(IIa), Formula (IIc), Formula (III), Formula (IIIa), Formula (IIIb),Formula (IIIc), Formula (IIId), Formula (IIIe), or Formula (IV), whereinthe “one or more linkers” referenced in the box of the foregoingformulas comprises a structure selected from the group consisting of:

wherein each linker is independent. In some embodiments of Formula (I),Formula (Ia), Formula (Ib), Formula (II), Formula (IIa), Formula (IIb),Formula (IIc), Formula (III), Formula (IIIa), Formula (IIIb), Formula(IIIc), Formula (IIId), Formula (IIIe), or Formula (IV), wherein each ofthe linkers independently has a structure of-(L¹)_(k1)-(L²)_(k2)-(L³)_(k3)-(L⁴)_(k4)-, wherein each of k1, k2, k3,and k4 is independently 0, 1 or 2, and each of the L¹, L², L³ and L⁴ isindependently selected from oxo, ester, amide, amino, C₁-C₃ alkylene,and —(CH₂—CH₂—O)₁₋₃—. In some embodiments, the sum of k1, k2, k3, and k4is an integer larger than or equal to 1. In some embodiments, the sum ofk1, k2, k3, and k4 is an integer larger than or equal to 2. As one ofordinary skill in the art would recognize “N” references nitrogen and “

” implies an attachment point.

In some embodiments of Formula (I), Formula (Ia), Formula (Ib), Formula(II), Formula (IIa), Formula (IIb), Formula (IIc), Formula (III),Formula (IIIa), Formula (IIIb), Formula (IIIc), Formula (IIId), Formula(IIIe), or Formula (IV), wherein each of the linkers independently has astructure of -(L¹)_(k1)-(L²)_(k2)-(L³)_(k3)-(L⁴)_(k4)-, wherein each ofk1, k2, k3, and k4 is independently 0, 1 or 2, and each of the L¹, L²,L³ and L⁴ is independently selected from —O—, —S—, S(═O)₁₋₂—, —C(═O)—,—C(═S)—, —NR^(L)—, —OC(═O)—, —C(═O)O—, —OC(═O)O—, —C(═O)NR^(L)—,—OC(═O)NR^(L)—, —NR^(L)C(═O)—, —NR^(L)C(═O) NR^(L)—, —P(═O)R^(L)—,—NR^(L)S(═O)(═NR^(L))—, —NR^(L)S(═O)₂—, —S(═O)₂NR^(L)—, —N═N—,—(CH₂—CH₂—O)₁₋₆—, linear or branched C₁₋₆ alkylene, linear or branchedC₂₋₆ alkenylene, linear or branched C₂₋₆ alkynylene, C₃-C₈cycloalkylene, C₂-C₇ heterocycloalkylene, C₆-C₁₀ arylene, and C₅-C₉heteroarylene, wherein the alkylene, alkenylene, alkynylene,cycloalkylene, cycloalkylene, arylene, or heteroarylene is substitutedor unsubstituted, and wherein each R^(L) is independently H, D, cyano,halogen, substituted or unsubstituted C₁-C₆ alkyl, —CD₃, —OCD₃,substituted or unsubstituted C₁-C₆ haloalkyl, substituted orunsubstituted C₁-C₆ heteroalkyl, substituted or unsubstitutedC₃-C₈cycloalkyl, substituted or unsubstituted C₂-C₇ heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl. In some embodiments, each R^(L) is independently H,substituted or unsubstituted C₁-C₆ alkyl, —OCH₃, substituted orunsubstituted C₁-C₆ haloalkyl, substituted or unsubstituted C₁-C₆heteroalkyl, substituted or unsubstituted C₃-C₈cycloalkyl, orsubstituted or unsubstituted C₂-C₇ heterocycloalkyl.

In some embodiments of Formula (I), Formula (Ia), Formula (Ib), Formula(II), Formula (IIa), Formula (IIb), Formula (IIc), Formula (III),Formula (IIIa), Formula (IIIb), Formula (IIIc), Formula (IIId), Formula(IIIe), or Formula (IV), each of the linkers independently comprises astructure selected from:

wherein each of the p, q, m, and n is independently 0, 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 14, 13, 15, 16, 17, 18, 19, or 20. In someembodiments, each of the p, q, m, and n is independently 0, 1, 2, 3, 4,or 5.

In some embodiments of Formula (I), Formula (Ia), Formula (Ib), Formula(II), Formula (IIa), Formula (IIb), Formula (IIc), Formula (III),Formula (IIIa), Formula (IIIb), Formula (IIIc), Formula (IIId), Formula(IIIe), or Formula (IV), wherein the “one or more linkers” referenced inthe box of the foregoing formulas comprises a structure that is

wherein each of the p, q, m, and n is independently 0, 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 14, 13, 15, 16, 17, 18, 19, or 20. In someembodiments, each of the p, q, m, and n is independently 0, 1, 2, 3, 4,or 5.

In some embodiments of Formula (I), Formula (Ia), Formula (Ib), Formula(II), Formula (IIa), Formula (IIb), Formula (IIc), Formula (III),Formula (IIIa), Formula (IIIb), Formula (IIIc), Formula (IIId), Formula(IIIe), or Formula (IV), W comprises one or more modified DNA or RNAbases. The nucleobases can comprise any chemical modifications asdescribed herein. In some embodiments, the nucleobases include a 2′-OHor 2′-OMe modification. For example, W may comprise one or more 2′-OMemodified adenine, cytosine, guanidine and uracil, referred to as (a)(c), (g), or (u). In some embodiments, a modified RNA, e.g. a gRNA ormRNA, includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 20, 25, 30, 35, 40, 50 or more modified nucleobases. In someembodiments, a modified RNA comprises one or more modified nucleobasesnear the 5′ end, near the 3′ end, or in the middle of the sequence. Themodified nucleobases within a modified RNA may or may not be contiguous.In some embodiments, a modified RNA comprises one or more 2′-OMemodifications scattered along the length of the sequence. In someembodiments, a modified RNA comprises one or more 2′OH modificationsscattered along the length of the sequence. In some embodiments, amodified RNA comprises alternating 2′-OH and 2′OMe modifications. Insome embodiments, W comprises (A)n, (T)n, (U)n, (a)n, or (u)n, wherein nis an integer no less than 3, wherein a is 2′-O-methyladenosine (2′-OMeA), and wherein u is 2′-O-methyluridine (2′-OMe-U). In some embodiments,W comprises (u)n, wherein n is an integer from 3 to 50 (SEQ ID NO: 118).In some embodiments, W comprises (u)n, wherein n is an integer from 3 to20 (SEQ ID NO: 119) or 3 to 15 (SEQ ID NO: 120). In some embodiments, Wcomprises one or more nucleotide sequences that are complementary to acoupling sequence. In some embodiments, W comprises one or more guaninesor cytidines. In some embodiments, W comprises at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50 or moreguanines or cytidines. In some embodiments, the one or more guanines orcytidines are complementary to one or more cytindines or guanines in acoupling sequence. In some embodiments, the guanines or cytidines are atthe terminals of W or the coupling sequence. Not intended to be bound byany theory, it is contemplated that the guaninies-cytidine pairing forms“GC locks” or “CG locks” that would increase binding affinity. Theguanines and/or cytidines in W or a coupling sequence may or may not becontiguous and may comprise any one of the chemical modifications asdescribed herein, e.g. a 2′-OMe or 2′-OH modification.

Receptor Targeting Conjugates

The key to fulfilling of nucleic acid-based therapy is the safe andefficacious delivery of payload to specific cell types and tissues.Lipid nanoparticles (LNPs) represent the most advanced non-viral drugdelivery technological platforms in the present time. LNPs arephysically able to pass through blood vessels and reach hepatocytes [Am.J. Pathol. 2010, 176, 14-21]. It has also been revealed thatapolipoprotein E (ApoE) proteins bind to the LNPs post PEG-lipiddiffusion from the LNP surface with a near neutral charge in the bloodstream, and function as an endogenous ligand against hepatocytes, whichexpress the low-density lipoprotein receptor (LDLr) [Mol. Ther., 2010,18, 1357-1364.]. It is accordingly envisioned that two key factors thatcontrol the efficient hepatic delivery of LNP are: 1) effectivePEG-lipid shedding from LNP surface in blood serum and 2) ApoE bindingto the LNP. The above endogenous ApoE-mediated LDLr-dependent LNPdelivery route is not an effective path to achieve LNP-based hepaticgene delivery for the LDLr deficient patient population.

In one aspect, described herein are LNPs comprising receptor targetingconjugates. In some aspects, described herein are receptor targetingconjugates. The LNPs with targeting conjugates are constituted to havethe receptor targeting moiety on the surface or periphery of theparticle. In one aspect low mol % of the receptor targeting conjugate isused while constituting the targeting LNP to achieve low surface densityof the targeting moiety on the surface/peripherry of the particle. Inanother aspect, high mol % of the receptor targeting conjugate is usedwhile constituting the targeting LNP to achieve high surface density ofthe targeting moiety on the surface/peripherry of the particle. Inanother aspect, desired mol % of the receptor targeting conjugate isused to achieve a range of surface density of the targeting moiety onthe surface/peripherry of the particle. In some embodiments, thereceptor targeting conjugate comprises a targeting moiety (or ligand), alinker, and a lipophilic moiety that is connected to the targetingmoiety. In some embodiments, the receptor targeting moiety (or ligand)targets a lectin receptor. In some embodiments, the lectin receptor isasialoglycoprotein receptor (ASGPR). In some embodiments the receptortargeting moiety is GalNAc or a derivative GalNAc that targets ASGPR. Inone aspect the receptor targeting conjugate comprises of one GalNAcmoiety or derivative thereof. In another aspect, the receptor targetingconjugate comprises of two GalNAc moieties or derivative thereof. Inanother aspect, the receptor targeting conjugate comprises of threeGalNAc moieties or derivate thereof. In another aspect, the receptortargeting conjugate is lipophilic. In some embodiments, the receptortargeting conjugate comprises one or more GalNAc moieties and one ormore lipid moieties, i.e., GalNAc-Lipid. In some embodiments, thereceptor targeting conjugate is a GalNAc-Lipid.

The current disclosure provides tissue specific efficient LNP deliveryto hepatocytes in an LDLr independent manner. The developed by thepresent disclosure trivalent GalNAc-moieties are attached to hydrophobicglycerol-based dialkyl lipids chain, sterol (cholesterol, for e.g.) andhydrophobic α-tocopherol through different PEG-spacers. These GalNAcconjugated lipids are then formulated with various excipients to yieldLNPs that carry low to high surface density of the custom-designedGalNAc ligands to target the asialoglycoprotein receptor (ASGPR), whichis highly expressed on the surface of hepatocytes.

The ligand on the surface of the engineered LNPs facilitatesASGPR-mediated tissue-specific uptake into hepatocytes. DifferentGalNAc-LNPs are constituted to circumvent ApoE biding and to enableGalNAc-ASGPR interaction to facilitate clathrin-mediated uptake intohepatocytes. Modulating PEG-shedding kinetics and modulating net surfacecharge density of GalNAc-LNP particles by using PEG-lipids describedherein in combination with GalNAc-lipids with varying PEG-tethers yieldGalNAc-LNPs that lack endogenous ApoE-binding characteristics to deliverparticles that carry RNA-payloads specifically to hepatocytes ofLDLR-deficient preclinical animal models at safe and efficacious dose.Dose-optimization in pre-clinical animal models further advance leadGalNAc-LNP (or LNPs) to clinical development to treat LDLR-deficientpatient population to elicit genome-editing at therapeutically viablesafe and efficacious dose.

Accordingly, in one aspect, disclosed herein is a receptor targetingconjugate, comprising a compound of Formula (V):

-   -   wherein,    -   a plurality of the A groups collectively comprise a receptor        targeting ligand;    -   each of L¹, L², L³, L⁴, L⁵, L⁶, L⁷, L⁸, L⁹, L¹⁰ and L¹² is        independently substituted or unsubstituted C₁-C₁₂ alkylene,        substituted or unsubstituted C₁-C₁₂ heteroalkylene, substituted        or unsubstituted C₂-C₁₂ alkenylene, substituted or unsubstituted        C₂-C₁₂ alkynylene, —(CH₂CH₂O)_(m)—, —(OCH₂CH₂)_(m)—, —O—, —S—,        —S(═O)—, —S(═O)₂—, —S(═O)(═NR¹)—, —C(═O)—, —C(═N—OR¹)—,        —C(═O)O—, —OC(═O)—, —C(═O)C(═O)—, —C(═O)NR¹—, —NR¹C(═O)—,        —OC(═O)NR¹—, —NR¹C(═O)O—, —NR¹C(═O)NR¹—, —C(═O)NR¹C(═O)—,        —S(═O)₂NR¹—, —NR¹S(═O)₂—, —NR¹—, —N(OR¹)—, —O[(P═O)O⁻]O— or        —O[(P═O)S⁻]O— or a bond;    -   L¹¹ is substituted or unsubstituted —(CH₂CH₂O)_(n)—, substituted        or unsubstituted —(OCH₂CH₂)_(n)—, substituted or unsubstituted        —(CH₂)_(n)—, or a bond;    -   each R¹ is independently H or substituted or unsubstituted        C₁-C₆alkyl;    -   R is a lipid, nucleic acid, amino acid, protein, or lipid        nanoparticle;    -   m is an integer selected from 1 to 10; and    -   n is an integer selected from 1 to 200.

In some embodiments, a receptor targeting conjugate comprises a compoundof Formula (V):

-   -   wherein,    -   a plurality of the A groups collectively comprise a receptor        targeting ligand;    -   each of L¹, L², L³, L⁴, L⁵, L⁶, L⁷, L⁸, L⁹, L¹⁰ and L¹² is        independently substituted or unsubstituted C₁-C₁₂ alkylene,        substituted or unsubstituted C₁-C₁₂ heteroalkylene, substituted        or unsubstituted C₂-C₁₂ alkenylene, substituted or unsubstituted        C₂-C₁₂ alkynylene, —(CH₂CH₂O)_(m)—, —(OCH₂CH₂)_(m)—, —O—, —S—,        —S(═O)—, —S(═O)₂—, —S(═O)(═NR¹)—, —C(═O)—, —C(═N—OR¹)—,        —C(═O)O—, —OC(═O)—, —C(═O)C(═O)—, —C(═O)NR¹—, —NR¹C(═O)—,        —OC(═O)NR¹—, —NR¹C(═O)O—, —NR¹C(═O)NR¹—, —C(═O)NR¹C(═O)—,        —S(═O)₂NR¹—, —NR¹S(═O)₂—, —NR¹—, or —N(OR¹)—;    -   L¹¹ is substituted or unsubstituted —(CH₂CH₂O)_(n)—, or        substituted or unsubstituted —(OCH₂CH₂)_(n)—;    -   each R¹ is independently H or substituted or unsubstituted        C₁-C₆alkyl;    -   R is a lipid, nucleic acid, amino acid, protein, or lipid        nanoparticle;    -   m is an integer selected from 1 to 10; and    -   n is an integer selected from 1 to 200.

In some embodiments, L¹¹ is —(CH₂CH₂O)_(n)— or —(OCH₂CH₂)_(n)—.

In some embodiments of a compound of Formula (V), A binds to a lectin.In some embodiment, the lectin is an asialoglycoprotein receptor(ASGPR). In some embodiments, A comprises one or moreN-acetylgalactosamine (GalNAc) or GalNAc derivatives.

In some embodiments of a compound of Formula (V), A isN-acetylgalactosamine (GalNAc) or a derivative thereof. In someembodiments, A is GalNAc. In some embodiments, A is or comprisesgalactose.

In some embodiments of a compound of Formula (V), each L¹, L⁴, and L⁷ isindependently substituted or unsubstituted C₁-C₁₂ alkylene. In someembodiments of a compound of Formula (V), each L¹, L⁴, and L⁷ isindependently substituted or unsubstituted C₂-C₆ alkylene. In someembodiments of a compound of Formula (V), each L¹, L⁴, and L⁷ is C₄alkylene.

In some embodiments of a compound of Formula (V), each L², L⁵, and L⁸ isindependently —C(═O)NR¹—, —NR¹C(═O)—, —OC(═O)NR¹—, —NR¹C(═O)O—,—NR¹C(═O)NR¹—, or —C(═O)NR¹C(═O)—. In some embodiments of a compound ofFormula (V), each L², L⁵, and L⁸ is independently —C(═O)NR¹— or—NR¹C(═O)—. In some embodiments of a compound of Formula (V), each L²,L⁵, and L⁸ is —C(═O)NH—.

In some embodiments of a compound of Formula (V), each L³, L⁶, and L⁹ isindependently substituted or unsubstituted C₁-C₁₂ alkylene. In someembodiments of a compound of Formula (V), each L³ is substituted orunsubstituted C₂-C₆ alkylene. In some embodiments of a compound ofFormula (V), L³ is C₄ alkylene. In some embodiments of a compound ofFormula (V), each L⁶ and L⁹ is independently substituted orunsubstituted C₂-C₁₀ alkylene. In some embodiments of a compound ofFormula (V), each L⁶ and L⁹ is independently substituted orunsubstituted C₂-C₆ alkylene. In some embodiments of a compound ofFormula (V), each L⁶ and L⁹ is C₃ alkylene.

In some embodiments of a compound of Formula (V), R¹ is H. In someembodiments, R¹ is substituted or unsubstituted C₁-C₆alkyl. In someembodiments, R¹ is methyl.

In another aspect, disclosed herein is a receptor targeting conjugate,comprising a compound of Formula (VI):

-   -   wherein,    -   a plurality of the A groups collectively comprise a receptor        targeting ligand;    -   each of L¹, L², L³, L⁴, L⁵, L⁶, L⁷, L⁸, L⁹, L¹⁰ and L¹² is        independently substituted or unsubstituted C₁-C₁₂ alkylene,        substituted or unsubstituted C₁-C₁₂ heteroalkylene, substituted        or unsubstituted C₂-C₁₂ alkenylene, substituted or unsubstituted        C₂-C₁₂ alkynylene, —(CH₂CH₂O)_(m)—, —(OCH₂CH₂)_(m)—, —O—, —S—,        —S(═O)—, —S(═O)₂—, —S(═O)(═NR¹)—, —C(═O)—, —C(═N—OR¹)—,        —C(═O)O—, —OC(═O)—, —C(═O)C(═O)—, —C(═O)NR¹—, —NR¹C(═O)—,        —OC(═O)NR¹—, —NR¹C(═O)O—, —NR¹C(═O)NR¹—, —C(═O)NR¹C(═O)—,        —S(═O)₂NR¹—, —NR¹S(═O)₂—, —NR¹—, —N(OR¹)—, —O[(P═O)O⁻]O—,        —O[(P═O)S⁻]O—, or a bond;    -   L¹¹ is substituted or unsubstituted —(CH₂CH₂O)_(n)—, substituted        or unsubstituted —(OCH₂CH₂)_(n)—, substituted or unsubstituted        —(CH₂)_(n)—, or a bond;    -   each R¹ is independently H or substituted or unsubstituted        C₁-C₆alkyl;    -   R is a lipid, nucleic acid, amino acid, protein, or lipid        nanoparticle;    -   m is an integer selected from 1 to 10; and    -   n is an integer selected from 1 to 200.

In some embodiments, disclosed herein is a receptor targeting conjugate,comprising a compound of Formula (VI):

-   -   wherein,    -   a plurality of the A groups collectively comprise a receptor        targeting ligand;    -   each of L¹, L², L³, L⁴, L⁵, L⁶, L⁷, L⁸, L⁹, L¹⁰ and L¹² is        independently substituted or unsubstituted C₁-C₁₂ alkylene,        substituted or unsubstituted C₁-C₁₂ heteroalkylene, substituted        or unsubstituted C₂-C₁₂ alkenylene, substituted or unsubstituted        C₂-C₁₂ alkynylene, —(CH₂CH₂O)_(m)—, —(OCH₂CH₂)_(m)—, —O—, —S—,        —S(═O)—, —S(═O)₂—, —S(═O)(═NR¹)—, —C(═O)—, —C(═N—OR¹)—,        —C(═O)O—, —OC(═O)—, —C(═O)C(═O)—, —C(═O)NR¹—, —NR¹C(═O)—,        —OC(═O)NR¹—, —NR¹C(═O)O—, —NR¹C(═O)NR¹—, —C(═O)NR¹C(═O)—,        —S(═O)₂NR¹—, —NR¹S(═O)₂—, —NR¹—, or —N(OR¹)—;    -   L¹¹ is substituted or unsubstituted —(CH₂CH₂O)_(n)— or        substituted or unsubstituted —(OCH₂CH₂)_(n)—;    -   each R¹ is independently H or substituted or unsubstituted        C₁-C₆alkyl;    -   R is a lipid, nucleic acid, amino acid, protein, or lipid        nanoparticle;    -   m is an integer selected from 1 to 10; and    -   n is an integer selected from 1 to 200.

In some embodiments, L¹¹ is —(CH₂CH₂O)_(n)— or —(OCH₂CH₂)_(n)—.

In some embodiments of a compound of Formula (VI), A binds to a lectin.In some embodiment, the lectin is an asialoglycoprotein receptor(ASGPR). In some embodiments, A comprises one or moreN-acetylgalactosamine (GalNAc) or GalNAc derivatives.

In some embodiments of a compound of Formula (VI), A isN-acetylgalactosamine (GalNAc) or a derivative thereof. In someembodiments, A is GalNAc.

In some embodiments of a compound of Formula (VI), each L¹, L⁴, and L⁷is independently substituted or unsubstituted C₁-C₁₂ alkylene orsubstituted or unsubstituted C₁-C₁₂ heteroalkylene.

In some embodiments of a compound of Formula (VI), each L¹, L⁴, and L⁷is independently substituted or unsubstituted C₁-C₁₂ heteroalkylene.

In some embodiments of a compound of Formula (VI), each L¹, L⁴, and L⁷is independently substituted or unsubstituted C₁-C₁₂ heteroalkylenecomprising 1-10 O atoms.

In some embodiments of a compound of Formula (VI), each L¹, L⁴, and L⁷is independently —(CH₂CH₂O)_(p1)—(CH₂)_(q1)—; wherein p1 is 1-8; and q1is 1-6.

In some embodiments of a compound of Formula (VI), each L¹, L⁴, and L⁷is —(CH₂CH₂O)₃—(CH₂)₂—.

In some embodiments of a compound of Formula (VI), each L¹, L⁴, and L⁷is independently substituted or unsubstituted C₁-C₁₂ alkylene.

In some embodiments of a compound of Formula (VI), each L¹, L⁴, and L⁷is independently substituted or unsubstituted C₂-C₆ alkylene.

In some embodiments of a compound of Formula (VI), each L¹, L⁴, and L⁷is C₄ alkylene.

In some embodiments of a compound of Formula (VI), each L², L⁵, and L⁸is independently —C(═O)NR¹—, —NR¹C(═O)—, —OC(═O)NR¹—, —NR¹C(═O)O—,—NR¹C(═O)NR¹—, or —C(═O)NR¹C(═O)—.

In some embodiments of a compound of Formula (VI), each L², L⁵, and L⁸is independently —C(═O)NR¹— or —NR¹C(═O)—.

In some embodiments of a compound of Formula (VI), each L², L⁵, and L⁸is —NHC(═O)—.

In some embodiments of a compound of Formula (VI), each L², L⁵, and L⁸is —C(═O)NH—.

In some embodiments of a compound of Formula (VI), each L³, L⁶, and L⁹is independently substituted or unsubstituted C₁-C₁₂ heteroalkylene.

In some embodiments of a compound of Formula (VI), each L³, L⁶, and L⁹is independently substituted or unsubstituted C₁-C₁₂ heteroalkylenecomprising 1-10 O atoms.

In some embodiments of a compound of Formula (VI), each L³, L⁶, and L⁹is independently —(CH₂CH₂O)_(p2)—(CH₂CH₂CH₂O)_(q2)—; wherein p2 is 1-8;and q2 is 1-6. In some embodiments, p2 is 1. In some embodiments, p2 is2. In some embodiments, p2 is 3. In some embodiments, p2 is 4. In someembodiments, p2 is 5. In some embodiments, p2 is 6. In some embodiments,p2 is 7. In some embodiments, p2 is 8. In some embodiments, q2 is 1. Insome embodiments, q2 is 2. In some embodiments, q2 is 3. In someembodiments, q2 is 4. In some embodiments, q2 is 5. In some embodiments,q2 is 6.

In some embodiments of a compound of Formula (VI), each L³, L⁶, and L⁹is —(CH₂CH₂O)—(CH₂CH₂CH₂O)—.

In some embodiments of a compound of Formula (VI), each L³, L⁶, and L⁹is independently —(CH₂CH₂CH₂O)_(q1)—; wherein q3 is 1-8. In someembodiments, q3 is 1. In some embodiments, q3 is 2. In some embodiments,q3 is 3. In some embodiments, q3 is 4. In some embodiments, q3 is 5. Insome embodiments, q3 is 6. In some embodiments, q3 is 7. In someembodiments, q3 is 8.

In some embodiments of a compound of Formula (VI), each L³, L⁶, and L⁹is —(CH₂CH₂CH₂O)₂—.

In some embodiments, a compound of Formula (VI) has a structure ofFormula (VIa):

wherein

each q4 is 1-10.

In some embodiments of a compound of Formula (VIb), q4 is 1-8. In someembodiments, q4 is 1-4. In some embodiments, q4 is 1-3. In someembodiments, q4 is 1. In some embodiments, q4 is 2. In some embodiments,q4 is 3. In some embodiments, q4 is 4. In some embodiments, q4 is 5.

In some embodiments of a compound of Formula (V) or Formula (VI), L¹⁰ issubstituted or unsubstituted C₁-C₁₂ alkylene. In some embodiments, L¹⁰is substituted or unsubstituted C₁-C₄ alkylene. In some embodiments, L¹⁰is C₂ alkylene.

In some embodiments, a compound of Formula (VI) has a structure ofFormula (VIb):

wherein,

r is 1-4.

In some embodiments of a compound of Formula (VIb), r is 1, 2, or 3. Insome embodiments, r is 1 or 2. In some embodiments, r is 2 or 3. In someembodiments, r is 1. In some embodiments, r is 2. In some embodiments, ris 3. In some embodiments, r is 4.

In some embodiments of a compound of Formula (V), Formula (VI), Formula(VIa), or Formula (VIb), L¹¹ is —(OCH₂CH₂)_(n)—. In some embodiments, nis 1-100. In some embodiments, n is 2-50. In some embodiments, n is10-50. In some embodiments, n is 20-50. In some embodiments, n is 30-50.In some embodiments, n is 40-50. In some embodiments, n is 2, 12, 37, or45. In some embodiments, n is 1. In some embodiments, n is 2. In someembodiments, n is 3. In some embodiments, n is 4. In some embodiments, nis 5. In some embodiments, n is 6. In some embodiments, n is 7. In someembodiments, n is 8. In some embodiments, n is 9. In some embodiments, nis 10. In some embodiments, n is 11. In some embodiments, n is 12. Insome embodiments, n is 13. In some embodiments, n is 14. In someembodiments, n is 15. In some embodiments, n is 16. In some embodiments,n is 17. In some embodiments, n is 18. In some embodiments, n is 19. Insome embodiments, n is 20. In some embodiments, n is 21. In someembodiments, n is 22. In some embodiments, n is 23. In some embodiments,n is 24. In some embodiments, n is 25. In some embodiments, n is 26. Insome embodiments, n is 27. In some embodiments, n is 28. In someembodiments, n is 29. In some embodiments, n is 30. In some embodiments,n is 31. In some embodiments, n is 32. In some embodiments, n is 33. Insome embodiments, n is 34. In some embodiments, n is 35. In someembodiments, n is 36. In some embodiments, n is 37. In some embodiments,n is 38. In some embodiments, n is 39. In some embodiments, n is 40. Insome embodiments, n is 41. In some embodiments, n is 42. In someembodiments, n is 43. In some embodiments, n is 44. In some embodiments,n is 45. In some embodiments, n is 46. In some embodiments, n is 47. Insome embodiments, n is 48. In some embodiments, n is 49. In someembodiments, n is 50. In some embodiments, n is at least 1. In someembodiments, n is at least 2. In some embodiments, n is at least 3. Insome embodiments, n is at least 4. In some embodiments, n is at least 5.In some embodiments, n is at least 6. In some embodiments, n is at least7. In some embodiments, n is at least 8. In some embodiments, n is atleast 9. In some embodiments, n is at least 10. In some embodiments, nis at least 11. In some embodiments, n is at least 12. In someembodiments, n is at least 13. In some embodiments, n is at least 14. Insome embodiments, n is at least 15. In some embodiments, n is at least16. In some embodiments, n is at least 17. In some embodiments, n is atleast 18. In some embodiments, n is at least 19. In some embodiments, nis at least 20. In some embodiments, n is at least 21. In someembodiments, n is at least 22. In some embodiments, n is at least 23. Insome embodiments, n is at least 24. In some embodiments, n is at least25. In some embodiments, n is at least 26. In some embodiments, n is atleast 27. In some embodiments, n is at least 28. In some embodiments, nis at least 29. In some embodiments, n is at least 30. In someembodiments, n is at least 31. In some embodiments, n is at least 32. Insome embodiments, n is at least 33. In some embodiments, n is at least34. In some embodiments, n is at least 35. In some embodiments, n is atleast 36. In some embodiments, n is at least 37. In some embodiments, nis at least 38. In some embodiments, n is at least 39. In someembodiments, n is at least 40. In some embodiments, n is at least 41. Insome embodiments, n is at least 42. In some embodiments, n is at least43. In some embodiments, n is at least 44. In some embodiments, n is atleast 45. In some embodiments, n is at least 46. In some embodiments, nis at least 47. In some embodiments, n is at least 48. In someembodiments, n is at least 49. In some embodiments, n is at most 2. Insome embodiments, n is at most 3. In some embodiments, n is at most 4.In some embodiments, n is at most 5. In some embodiments, n is at most6. In some embodiments, n is at most 7. In some embodiments, n is atmost 8. In some embodiments, n is at most 9. In some embodiments, n isat most 10. In some embodiments, n is at most 11. In some embodiments, nis at most 12. In some embodiments, n is at most 13. In someembodiments, n is at most 14. In some embodiments, n is at most 15. Insome embodiments, n is at most 16. In some embodiments, n is at most 17.In some embodiments, n is at most 18. In some embodiments, n is at most19. In some embodiments, n is at most 20. In some embodiments, n is atmost 21. In some embodiments, n is at most 22. In some embodiments, n isat most 23. In some embodiments, n is at most 24. In some embodiments, nis at most 25. In some embodiments, n is at most 26. In someembodiments, n is at most 27. In some embodiments, n is at most 28. Insome embodiments, n is at most 29. In some embodiments, n is at most 30.In some embodiments, n is at most 31. In some embodiments, n is at most32. In some embodiments, n is at most 33. In some embodiments, n is atmost 34. In some embodiments, n is at most 35. In some embodiments, n isat most 36. In some embodiments, n is at most 37. In some embodiments, nis at most 38. In some embodiments, n is at most 39. In someembodiments, n is at most 40. In some embodiments, n is at most 41. Insome embodiments, n is at most 42. In some embodiments, n is at most 43.In some embodiments, n is at most 44. In some embodiments, n is at most45. In some embodiments, n is at most 46. In some embodiments, n is atmost 47. In some embodiments, n is at most 48. In some embodiments, n isat most 49. In some embodiments, n is at most 50.

In some embodiments of a compound of Formula (V), Formula (VI), Formula(VIa), or Formula (VIb), L¹² is —O—, —C(═O)O—, —C(═O)NR¹—, —NR¹C(═O)—,or —NR¹C(═O)O—. In some embodiments, L¹² is —C(═O)O— or —NR¹C(═O)O—. Insome embodiments, L¹² is —C(═O)O—. In some embodiments, L¹² is—NHC(═O)O—. In some embodiments, L¹² is —NHC(═O)—.

In some embodiments of a compound of Formula (VI), Formula (VIa), orFormula (VIb), R¹ is H. In some embodiments, leis substituted orunsubstituted C₁-C₆alkyl. In some embodiments, R¹ is methyl.

In accordance with the foregoing referenced formulas, in someembodiments of a compound of Formula (V) or (VI), L¹ is substituted orunsubstituted C₁-C₁₂ alkylene, substituted or unsubstituted C₁-C₁₂heteroalkylene, substituted or unsubstituted C₂-C₁₂ alkenylene,substituted or unsubstituted C₂-C₁₂ alkynylene, —(CH₂CH₂O)_(m)—,—(OCH₂CH₂)_(m)—, —O—, —S—, —S(═O)—, —S(═O)₂—, —S(═O)(═NR¹)—, —C(═O)—,—C(═N—OR¹)—, —C(═O)O—, —OC(═O)—, —C(═O)C(═O)—, —C(═O)NR¹—, —NR¹C(═O)—,—OC(═O)NR¹—, —NR¹C(═O)O—, —NR¹C(═O)NR¹—, —C(═O)NR¹C(═O)—, —S(═O)₂NR¹—,—NR¹S(═O)₂—, —NR^(I-)—, —N(OR¹)—, —O[(P═O)O⁻]O—, —O[(P═O)S⁻]O—, or abond. In some embodiments, L¹ is substituted or unsubstituted C₁-C₁₂alkylene. In some embodiments, L¹ is substituted or unsubstituted C₁-C₁₂heteroalkylene. In some embodiments, L¹ is substituted or unsubstitutedC₂-C₁₂ alkenylene. In some embodiments, L¹ is substituted orunsubstituted C₂-C₁₂ alkynylene. In some embodiments, L¹ is—(CH₂CH₂O)_(m)— or —(OCH₂CH₂)_(m)—. In some embodiments, L¹ is —O—. Insome embodiments, L¹ is —S—. In some embodiments, L¹ is —S(═O)—. In someembodiments, L¹ is —S(═O)₂—. In some embodiments, L¹ is —S(═O)(═NR¹)—.In some embodiments, L¹ is —C(═O)—. In some embodiments, L¹ is—C(═N—OR¹)—. In some embodiments, L¹ is —C(═O)O—. In some embodiments,L¹ is OC(═O)—. In some embodiments, L¹ is —C(═O)C(═O)—. In someembodiments, L¹ is —C(═O)NR¹—. In some embodiments, L¹ is —NR¹C(═O)—. Insome embodiments, L¹ is —OC(═O)NR¹—. In some embodiments, L¹ is—NR¹C(═O)O—. In some embodiments, L¹ is —NR¹C(═O)NR¹—. In someembodiments, L¹ is —C(═O)NR¹C(═O)—. In some embodiments, L¹ is—S(═O)₂NR¹—. In some embodiments, L¹ is —NR¹S(═O)₂—. In someembodiments, L¹ is —NR¹—. In some embodiments, L¹ is —N(OR¹)—. In someembodiments, L¹ is —O[(P═O)O⁻]O—. In some embodiments, L¹ is—O[(P═O)S⁻]O—. In some embodiments, L¹ is a bond.

In accordance with the foregoing referenced formulas, in someembodiments of a compound of Formula (V) or (VI), L² is substituted orunsubstituted C₁-C₁₂ alkylene, substituted or unsubstituted C₁-C₁₂heteroalkylene, substituted or unsubstituted C₂-C₁₂ alkenylene,substituted or unsubstituted C₂-C₁₂ alkynylene, —(CH₂CH₂O)_(m)—,—(OCH₂CH₂)_(m)—, —O—, —S—, —S(═O)—, —S(═O)₂—, —S(═O)(═NR¹)—, —C(═O)—,—C(═N—OR¹)—, —C(═O)O—, —OC(═O)—, —C(═O)C(═O)—, —C(═O)NR¹—, —NR¹C(═O)—,—OC(═O)NR¹—, —NR¹C(═O)O—, —NR¹C(═O)NR¹—, —C(═O)NR¹C(═O)—, —S(═O)₂NR¹—,—NR¹S(═O)₂—, —NR¹—, —N(OR¹)—, —O[(P═O)O⁻]O—, —O[(P═O)S⁻]O—, or a bond.In some embodiments, L² is substituted or unsubstituted C₁-C₁₂ alkylene.In some embodiments, L² is substituted or unsubstituted C₁-C₁₂heteroalkylene. In some embodiments, L² is substituted or unsubstitutedC₂-C₁₂ alkenylene. In some embodiments, L² is substituted orunsubstituted C₂-C₁₂ alkynylene. In some embodiments, L² is—(CH₂CH₂O)_(m)— or —(OCH₂CH₂)_(m)—. In some embodiments, L² is —O—. Insome embodiments, L² is —S—. In some embodiments, L² is —S(═O)—. In someembodiments, L² is —S(═O)₂—. In some embodiments, L² is —S(═O)(═NR¹)—.In some embodiments, L² is —C(═O)—. In some embodiments, L² is—C(═N—OR¹)—. In some embodiments, L² is —C(═O)O—. In some embodiments,L² is OC(═O)—. In some embodiments, L² is —C(═O)C(═O)—. In someembodiments, L² is —C(═O)NR¹—. In some embodiments, L² is —NR¹C(═O)—. Insome embodiments, L² is —NRHC(═O)—. In some embodiments, L² is—OC(═O)NR¹—. In some embodiments, L² is —NR¹C(═O)O—. In someembodiments, L² is —NR¹C(═O)NR¹—. In some embodiments, L² is—C(═O)NR¹C(═O)—. In some embodiments, L² is —S(═O)₂NR¹—. In someembodiments, L² is —NR¹S(═O)₂—. In some embodiments, L² is —NR¹—. Insome embodiments, L² is —N(OR¹)—. In some embodiments, L² is—O[(P═O)O⁻]O—. In some embodiments, L² is —O[(P═O)S⁻]O—. In someembodiments, L² is a bond.

In accordance with the foregoing referenced formulas, in someembodiments of a compound of Formula (V) or (VI), L³ is substituted orunsubstituted C₁-C₁₂ alkylene, substituted or unsubstituted C₁-C₁₂heteroalkylene, substituted or unsubstituted C₂-C₁₂ alkenylene,substituted or unsubstituted C₂-C₁₂ alkynylene, —(CH₂CH₂O)_(m)—,—(OCH₂CH₂)_(m)—, —O—, —S—, —S(═O)—, —S(═O)₂—, —S(═O)(═NR¹)—, —C(═O)—,—C(═N—OR¹)—, —C(═O)O—, —OC(═O)—, —C(═O)C(═O)—, —C(═O)NR¹—, —NR¹C(═O)—,—OC(═O)NR¹—, —NR¹C(═O)O—, —NR¹C(═O)NR¹—, —C(═O)NR¹C(═O)—, —S(═O)₂NR¹—,—NR¹S(═O)₂—, —NR¹—, —N(OR¹)—, —O[(P═O)O⁻]O—, —O[(P═O)S⁻]O—, or a bond.In some embodiments, L³ is substituted or unsubstituted C₁-C₁₂ alkylene.In some embodiments, L³ is an unsubstituted C₃₋₄ alkylene. In someembodiments, L³ is an unsubstituted C₁₋₄ alkylene. In some embodiments,L³ is substituted or unsubstituted C₁-C₁₂ heteroalkylene. In someembodiments, L³ is substituted or unsubstituted C₂-C₁₂ alkenylene. Insome embodiments, L³ is substituted or unsubstituted C₂-C₁₂ alkynylene.In some embodiments, L³ is —(CH₂CH₂O), or —(OCH₂CH₂)_(m)—. In someembodiments, L³ is —O—. In some embodiments, L³ is —S—. In someembodiments, L³ is —S(═O)—. In some embodiments, L³ is —S(═O)₂—. In someembodiments, L³ is —S(═O)(═NR¹)—. In some embodiments, L³ is —C(═O)—. Insome embodiments, L³ is —C(═N—OR¹)—. In some embodiments, L³ is—C(═O)O—. In some embodiments, L³ is OC(═O)—. In some embodiments, L³ is—C(═O)C(═O)—. In some embodiments, L³ is —C(═O)NR¹—. In someembodiments, L³ is —NR¹C(═O)—. In some embodiments, L³ is —OC(═O)NR¹—.In some embodiments, L³ is —NR¹C(═O)O—. In some embodiments, L³ is—NR¹C(═O)NR¹—. In some embodiments, L³ is —C(═O)NR¹C(═O)—. In someembodiments, L³ is —S(═O)₂NR¹—. In some embodiments, L³ is —NR¹S(═O)₂—.In some embodiments, L³ is —NR¹—. In some embodiments, L³ is —N(OR¹)—.In some embodiments, L³ is —O[(P═O)O⁻]O—. In some embodiments, L³ is—O[(P═O)S⁻]O—. In some embodiments, L³ is a bond.

In accordance with the foregoing referenced formulas, in someembodiments of a compound of Formula (V) or (VI), L⁴ is substituted orunsubstituted C₁-C₁₂ alkylene, substituted or unsubstituted C₁-C₁₂heteroalkylene, substituted or unsubstituted C₂-C₁₂ alkenylene,substituted or unsubstituted C₂-C₁₂ alkynylene, —(CH₂CH₂O)_(m)—,—(OCH₂CH₂)_(m)—, —O—, —S—, —S(═O)—, —S(═O)₂—, —S(═O)(═NR¹)—, —C(═O)—,—C(═N—OR¹)—, —C(═O)O—, —OC(═O)—, —C(═O)C(═O)—, —C(═O)NR¹—, —NR¹C(═O)—,—OC(═O)NR¹—, —NR¹C(═O)O—, —NR¹C(═O)NR¹—, —C(═O)NR¹C(═O)—, —S(═O)₂NR¹—,—NR¹S(═O)₂—, —NR¹—, —N(OR¹)—, —O[(P═O)O⁻]O—, —O[(P═O)S⁻]O—, or a bond.In some embodiments, L⁴ is substituted or unsubstituted C₁-C₁₂ alkylene.In some embodiments, L⁴ is an unsubstituted C₄ alkylene. In someembodiments, L⁴ is substituted or unsubstituted C₁-C₁₂ heteroalkylene.In some embodiments, L⁴ is substituted or unsubstituted C₂-C₁₂alkenylene. In some embodiments, L⁴ is substituted or unsubstitutedC₂-C₁₂ alkynylene. In some embodiments, L⁴ is —(CH₂CH₂O)_(m)— or—(OCH₂CH₂)_(m)—. In some embodiments, L⁴ is —O—. In some embodiments, L⁴is —S—. In some embodiments, L⁴ is —S(═O)—. In some embodiments, L⁴ is—S(═O)₂—. In some embodiments, L⁴ is —S(═O)(═NR¹)—. In some embodiments,L⁴ is —C(═O)—. In some embodiments, L⁴ is —C(═N—OR¹)—. In someembodiments, L⁴ is —C(═O)O—. In some embodiments, L⁴ is OC(═O)—. In someembodiments, L⁴ is —C(═O)C(═O)—. In some embodiments, L⁴ is —C(═O)NR¹—.In some embodiments, L⁴ is —NR¹C(═O)—. In some embodiments, L⁴ is—OC(═O)NR¹—. In some embodiments, L⁴ is —NR¹C(═O)O—. In someembodiments, L⁴ is —NR¹C(═O)NR¹—. In some embodiments, L⁴ is—C(═O)NR¹C(═O)—. In some embodiments, L⁴ is —S(═O)₂NR¹—. In someembodiments, L⁴ is —NR¹S(═O)₂—. In some embodiments, L⁴ is —NR¹—. Insome embodiments, L⁴ is —N(OR¹)—. In some embodiments, L⁴ is—O[(P═O)O⁻]O—. In some embodiments, L⁴ is —O[(P═O)S⁻]O—. In someembodiments, L⁴ is a bond.

In accordance with the foregoing referenced formulas, in someembodiments of a compound of Formula (V) or (VI), L⁵ is substituted orunsubstituted C₁-C₁₂ alkylene, substituted or unsubstituted C₁-C₁₂heteroalkylene, substituted or unsubstituted C₂-C₁₂ alkenylene,substituted or unsubstituted C₂-C₁₂ alkynylene, —(CH₂CH₂O)_(m)—,—(OCH₂CH₂)_(m)—, —O—, —S—, —S(═O)—, —S(═O)₂—, —S(═O)(═NR¹)—, —C(═O)—,—C(═N—OR¹)—, —C(═O)O—, —OC(═O)—, —C(═O)C(═O)—, —C(═O)NR¹—, —NR¹C(═O)—,—OC(═O)NR¹—, —NR¹C(═O)O—, —NR¹C(═O)NR¹—, —C(═O)NR¹C(═O)—, —S(═O)₂NR¹—,—NR¹S(═O)₂—, —NR¹—, —N(OR¹)—, —O[(P═O)O⁻]O—, —O[(P═O)S⁻]O—, or a bond.In some embodiments, L⁵ is substituted or unsubstituted C₁-C₁₂ alkylene.In some embodiments, L⁵ is substituted or unsubstituted C₁-C₁₂heteroalkylene. In some embodiments, L⁵ is substituted or unsubstitutedC₂-C₁₂ alkenylene. In some embodiments, L⁵ is substituted orunsubstituted C₂-C₁₂ alkynylene. In some embodiments, L⁵ is—(CH₂CH₂O)_(m)— or —(OCH₂CH₂)_(m)—. In some embodiments, L⁵ is —O—. Insome embodiments, L⁵ is —S—. In some embodiments, L⁵ is —S(═O)—. In someembodiments, L⁵ is —S(═O)₂—. In some embodiments, L⁵ is —S(═O)(═NR¹)—.In some embodiments, L⁵ is —C(═O)—. In some embodiments, L⁵ is—C(═N—OR¹)—. In some embodiments, L⁵ is —C(═O)O—. In some embodiments,L⁵ is OC(═O)—. In some embodiments, L⁵ is —C(═O)C(═O)—. In someembodiments, L⁵ is —C(═O)NR¹—. In some embodiments, L⁵ is —NR¹C(═O)—. Insome embodiments, L² is —NRHC(═O)—. In some embodiments, L⁵ is—OC(═O)NR¹—. In some embodiments, L⁵ is —NR¹C(═O)O—. In someembodiments, L⁵ is —NR¹C(═O)NR¹—. In some embodiments, L⁵ is—C(═O)NR¹C(═O)—. In some embodiments, L⁵ is —S(═O)₂NR¹—. In someembodiments, L⁵ is —NR¹S(═O)₂—. In some embodiments, L⁵ is —NR¹—. Insome embodiments, L⁵ is —N(OR¹)—. In some embodiments, L⁵ is—O[(P═O)O⁻]O—. In some embodiments, L⁵ is —O[(P═O)S⁻]O—. In someembodiments, L⁵ is a bond.

In accordance with the foregoing referenced formulas, in someembodiments of a compound of Formula (V) or (VI), L⁶ is substituted orunsubstituted C₁-C₁₂ alkylene, substituted or unsubstituted C₁-C₁₂heteroalkylene, substituted or unsubstituted C₂-C₁₂ alkenylene,substituted or unsubstituted C₂-C₁₂ alkynylene, —(CH₂CH₂O)_(m)—,—(OCH₂CH₂)_(m)—, —O—, —S—, —S(═O)—, —S(═O)₂—, —S(═O)(═NR¹)—, —C(═O)—,—C(═N—OR¹)—, —C(═O)O—, —OC(═O)—, —C(═O)C(═O)—, —C(═O)NR¹—, —NR¹C(═O)—,—OC(═O)NR¹—, —NR¹C(═O)O—, —NR¹C(═O)NR¹—, —C(═O)NR¹C(═O)—, —S(═O)₂NR¹—,—NR¹S(═O)₂—, —NR¹—, —N(OR¹)—, —O[(P═O)O⁻]O—, —O[(P═O)S⁻]O—, or a bond.In some embodiments, L⁶ is substituted or unsubstituted C₁-C₁₂ alkylene.In some embodiments, L⁶ is an unsubstituted C₃₋₄ alkylene. In someembodiments, L⁶ is an unsubstituted C₁₋₄ alkylene. In some embodiments,L⁶ is substituted or unsubstituted C₁-C₁₂ heteroalkylene. In someembodiments, L⁶ is substituted or unsubstituted C₂-C₁₂ alkenylene. Insome embodiments, L⁶ is substituted or unsubstituted C₂-C₁₂ alkynylene.In some embodiments, L⁶ is —(CH₂CH₂O), or —(OCH₂CH₂)_(m)—. In someembodiments, L⁶ is —O—. In some embodiments, L⁶ is —S—. In someembodiments, L⁶ is —S(═O)—. In some embodiments, L⁶ is —S(═O)₂—. In someembodiments, L⁶ is —S(═O)(═NR¹)—. In some embodiments, L⁶ is —C(═O)—. Insome embodiments, L⁶ is —C(═N—OR¹)—. In some embodiments, L⁶ is—C(═O)O—. In some embodiments, L⁶ is OC(═O)—. In some embodiments, L⁶ is—C(═O)C(═O)—. In some embodiments, L⁶ is —C(═O)NR¹—. In someembodiments, L⁶ is —NR¹C(═O)—. In some embodiments, L⁶ is —OC(═O)NR¹—.In some embodiments, L⁶ is —NR¹C(═O)O—. In some embodiments, L⁶ is—NR¹C(═O)NR¹—. In some embodiments, L⁶ is —C(═O)NR¹C(═O)—. In someembodiments, L⁶ is —S(═O)₂NR¹—. In some embodiments, L⁶ is —NR¹S(═O)₂—.In some embodiments, L⁶ is —NR¹—. In some embodiments, L⁶ is —N(OR¹)—.In some embodiments, L⁶ is —O[(P═O)O⁻]O—. In some embodiments, L⁶ is—O[(P═O)S⁻]O—. In some embodiments, L⁶ is a bond.

In accordance with the foregoing referenced formulas, in someembodiments of a compound of Formula (V) or (VI), L⁷ is substituted orunsubstituted C₁-C₁₂ alkylene, substituted or unsubstituted C₁-C₁₂heteroalkylene, substituted or unsubstituted C₂-C₁₂ alkenylene,substituted or unsubstituted C₂-C₁₂ alkynylene, —(CH₂CH₂O)_(m)—,—(OCH₂CH₂)_(m)—, —O—, —S—, —S(═O)—, —S(═O)₂—, —S(═O)(═NR¹)—, —C(═O)—,—C(═N—OR¹)—, —C(═O)O—, —OC(═O)—, —C(═O)C(═O)—, —C(═O)NR¹—, —NR¹C(═O)—,—OC(═O)NR¹—, —NR¹C(═O)O—, —NR¹C(═O)NR¹—, —C(═O)NR¹C(═O)—, —S(═O)₂NR¹—,—NR¹S(═O)₂—, —NR¹—, —N(OR¹)—, —O[(P═O)O⁻]O—, —O[(P═O)S⁻]O—, or a bond.In some embodiments, L⁷ is substituted or unsubstituted C₁-C₁₂ alkylene.In some embodiments, L⁷ is an unsubstituted C₄ alkylene. In someembodiments, L⁷ is substituted or unsubstituted C₁-C₁₂ heteroalkylene.In some embodiments, L⁷ is substituted or unsubstituted C₂-C₁₂alkenylene. In some embodiments, L⁷ is substituted or unsubstitutedC₂-C₁₂ alkynylene. In some embodiments, L⁷ is —(CH₂CH₂O)_(m)— or—(OCH₂CH₂)_(m)—. In some embodiments, L⁷ is —O—. In some embodiments, L⁷is —S—. In some embodiments, L⁷ is —S(═O)—. In some embodiments, L⁷ is—S(═O)₂—. In some embodiments, L⁷ is —S(═O)(═NR¹)—. In some embodiments,L⁷ is —C(═O)—. In some embodiments, L⁷ is —C(═N—OR¹)—. In someembodiments, L⁷ is —C(═O)O—. In some embodiments, L⁷ is OC(═O)—. In someembodiments, L⁷ is —C(═O)C(═O)—. In some embodiments, L⁷ is —C(═O)NR¹—.In some embodiments, L⁷ is —NR¹C(═O)—. In some embodiments, L⁷ is—OC(═O)NR¹—. In some embodiments, L⁷ is —NR¹C(═O)O—. In someembodiments, L⁷ is —NR¹C(═O)NR¹—. In some embodiments, L⁷ is—C(═O)NR¹C(═O)—. In some embodiments, L⁷ is —S(═O)₂NR¹—. In someembodiments, L⁷ is —NR¹S(═O)₂—. In some embodiments, L⁷ is —NR¹—. Insome embodiments, L⁷ is —N(OR¹)—. In some embodiments, L⁷ is—O[(P═O)O⁻]O—. In some embodiments, L⁷ is —O[(P═O)S⁻]O—. In someembodiments, L⁷ is a bond.

In accordance with the foregoing referenced formulas, in someembodiments of a compound of Formula (V) or (VI), L⁸ is substituted orunsubstituted C₁-C₁₂ alkylene, substituted or unsubstituted C₁-C₁₂heteroalkylene, substituted or unsubstituted C₂-C₁₂ alkenylene,substituted or unsubstituted C₂-C₁₂ alkynylene, —(CH₂CH₂O)_(m)—,—(OCH₂CH₂)_(m)—, —O—, —S—, —S(═O)—, —S(═O)₂—, —S(═O)(═NR¹)—, —C(═O)—,—C(═N—OR¹)—, —C(═O)O—, —OC(═O)—, —C(═O)C(═O)—, —C(═O)NR¹—, —NR¹C(═O)—,—OC(═O)NR¹—, —NR¹C(═O)O—, —NR¹C(═O)NR¹—, —C(═O)NR¹C(═O)—, —S(═O)₂NR¹—,—NR¹S(═O)₂—, —NR¹—, —N(OR¹)—, —O[(P═O)O⁻]O—, —O[(P═O)S⁻]O— or a bond. Insome embodiments, L⁸ is substituted or unsubstituted C₁-C₁₂ alkylene. Insome embodiments, L⁸ is substituted or unsubstituted C₁-C₁₂heteroalkylene. In some embodiments, L⁸ is substituted or unsubstitutedC₂-C₁₂ alkenylene. In some embodiments, L⁸ is substituted orunsubstituted C₂-C₁₂ alkynylene. In some embodiments, L⁸ is—(CH₂CH₂O)_(m)— or —(OCH₂CH₂)_(m)—. In some embodiments, L⁸ is —O—. Insome embodiments, L⁸ is —S—. In some embodiments, L⁸ is —S(═O)—. In someembodiments, L⁸ is —S(═O)₂—. In some embodiments, L⁸ is —S(═O)(═NR¹)—.In some embodiments, L⁸ is —C(═O)—. In some embodiments, L⁸ is—C(═N—OR¹)—. In some embodiments, L⁸ is —C(═O)O—. In some embodiments,L⁸ is OC(═O)—. In some embodiments, L⁸ is —C(═O)C(═O)—. In someembodiments, L⁸ is —C(═O)NR¹—. In some embodiments, L⁸ is —NR¹C(═O)—. Insome embodiments, L⁸ is —OC(═O)NR¹—. In some embodiments, L⁸ is—NR¹C(═O)O—. In some embodiments, L² is —NRHC(═O)—. In some embodiments,L⁸ is —NR¹C(═O)NR¹—. In some embodiments, L⁸ is —C(═O)NR¹C(═O)—. In someembodiments, L⁸ is —S(═O)₂NR¹—. In some embodiments, L⁸ is —NR¹S(═O)₂—.In some embodiments, L⁸ is —NR¹—. In some embodiments, L⁸ is —N(OR¹)—.In some embodiments, L⁸ is —O[(P═O)O⁻]O—. In some embodiments, L⁸ is—O[(P═O)S⁻]O—. In some embodiments, L⁸ is a bond.

In accordance with the foregoing referenced formulas, in someembodiments of a compound of Formula (V) or (VI), L⁹ is substituted orunsubstituted C₁-C₁₂ alkylene, substituted or unsubstituted C₁-C₁₂heteroalkylene, substituted or unsubstituted C₂-C₁₂ alkenylene,substituted or unsubstituted C₂-C₁₂ alkynylene, —(CH₂CH₂O)_(m)—,—(OCH₂CH₂)_(m)—, —O—, —S—, —S(═O)—, —S(═O)₂—, —S(═O)(═NR¹)—, —C(═O)—,—C(═N—OR¹)—, —C(═O)O—, —OC(═O)—, —C(═O)C(═O)—, —C(═O)NR¹—, —NR¹C(═O)—,—OC(═O)NR¹—, —NR¹C(═O)O—, —NR¹C(═O)NR¹—, —C(═O)NR¹C(═O)—, —S(═O)₂NR¹—,—NR¹S(═O)₂—, —NR¹—, —N(OR¹)—, —O[(P═O)O⁻]O—, —O[(P═O)S⁻]O—, or a bond.In some embodiments, L⁹ is substituted or unsubstituted C₁-C₁₂ alkylene.In some embodiments, L⁹ is an unsubstituted C₃₋₄ alkylene. In someembodiments, L⁹ is an unsubstituted C₁₋₄ alkylene. In some embodiments,L⁹ is substituted or unsubstituted C₁-C₁₂ heteroalkylene. In someembodiments, L⁹ is substituted or unsubstituted C₂-C₁₂ alkenylene. Insome embodiments, L⁹ is substituted or unsubstituted C₂-C₁₂ alkynylene.In some embodiments, L⁹ is —(CH₂CH₂O), or —(OCH₂CH₂)_(m)—. In someembodiments, L⁹ is —O—. In some embodiments, L⁹ is —S—. In someembodiments, L⁹ is —S(═O)—. In some embodiments, L⁹ is —S(═O)₂—. In someembodiments, L⁹ is —S(═O)(═NR¹)—. In some embodiments, L⁹ is —C(═O)—. Insome embodiments, L⁹ is —C(═N—OR¹)—. In some embodiments, L⁹ is—C(═O)O—. In some embodiments, L⁹ is OC(═O)—. In some embodiments, L⁹ is—C(═O)C(═O)—. In some embodiments, L⁹ is —C(═O)NR¹—. In someembodiments, L⁹ is —NR¹C(═O)—. In some embodiments, L⁹ is —OC(═O)NR¹—.In some embodiments, L⁹ is —NR¹C(═O)O—. In some embodiments, L⁹ is—NR¹C(═O)NR¹—. In some embodiments, L⁹ is —C(═O)NR¹C(═O)—. In someembodiments, L⁹ is —S(═O)₂NR¹—. In some embodiments, L⁹ is —NR¹S(═O)₂—.In some embodiments, L⁹ is —NR¹—. In some embodiments, L⁹ is —N(OR¹)—.In some embodiments, L⁹ is —O[(P═O)O⁻]O—. In some embodiments, L⁹ is—O[(P═O)S⁻]O—. In some embodiments, L⁹ is a bond.

In accordance with the foregoing referenced formulas, in someembodiments of a compound of Formula (V) or (VI), L¹⁰ is substituted orunsubstituted C₁-C₁₂ alkylene, substituted or unsubstituted C₁-C₁₂heteroalkylene, substituted or unsubstituted C₂-C₁₂ alkenylene,substituted or unsubstituted C₂-C₁₂ alkynylene, —(CH₂CH₂O)_(n)—,—(OCH₂CH₂)_(m)—, —O—, —S—, —S(═O)—, —S(═O)₂—, —S(═O)(═NR¹)—, —C(═O)—,—C(═N—OR¹)—, —C(═O)O—, —OC(═O)—, —C(═O)C(═O)—, —C(═O)NR¹—, —NR¹C(═O)—,—OC(═O)NR¹—, —NR¹C(═O)O—, —NR¹C(═O)NR¹—, —C(═O)NR¹C(═O)—, —S(═O)₂NR¹—,—NR¹S(═O)₂—, —NR¹—, —N(OR¹)—, —O[(P═O)O⁻]O—, —O[(P═O)S⁻]O—, or a bond.In some embodiments, L¹⁰ is substituted or unsubstituted C₁-C₁₂alkylene. In some embodiments, L¹⁰ is substituted or unsubstitutedC₁-C₁₂ heteroalkylene. In some embodiments, L¹⁰ is substituted orunsubstituted C₂-C₁₂ alkenylene. In some embodiments, L¹⁰ is substitutedor unsubstituted C₂-C₁₂ alkynylene. In some embodiments, L¹⁰ is—(CH₂CH₂O)_(m)— or —(OCH₂CH₂)_(m)—. In some embodiments, L¹⁰ is —O—. Insome embodiments, L¹⁰ is —S—. In some embodiments, L¹⁰ is —S(═O)—. Insome embodiments, L¹⁰ is —S(═O)₂—. In some embodiments, L¹⁰ is—S(═O)(═NR¹)—. In some embodiments, L¹⁰ is —C(═O)—. In some embodiments,L¹⁰ is —C(═N—OR¹)—. In some embodiments, L¹⁰ is —C(═O)O—. In someembodiments, L¹⁰ is OC(═O)—. In some embodiments, L¹⁰ is —C(═O)C(═O)—.In some embodiments, L¹⁰ is —C(═O)NR¹—. In some embodiments, L¹⁰ is—NR¹C(═O)—. In some embodiments, L¹⁰ is —OC(═O)NR¹—. In someembodiments, L¹⁰ is —NR¹C(═O)O—. In some embodiments, L¹⁰ is—NR¹C(═O)NR¹—. In some embodiments, L¹⁰ is —C(═O)NR¹C(═O)—. In someembodiments, L¹⁰ is —S(═O)₂NR¹—. In some embodiments, L¹⁰ is—NR¹S(═O)₂—. In some embodiments, L¹⁰ is —NR¹—. In some embodiments, L¹⁰is —N(OR¹)—. In some embodiments, L¹⁰ is —O[(P═O)O⁻]O—. In someembodiments, L¹⁰ is —O[(P═O)S⁻]O—. In some embodiments, L¹⁰ issubstituted or unsubstituted C₁-C₆ alkylene. In some embodiments, L¹⁰ issubstituted or unsubstituted C₁-C₃ alkylene. In some embodiments, L¹⁰ issubstituted or unsubstituted C₂-C₃ alkylene. In some embodiments, L¹⁰ is—CH₂CH₂—. In some embodiments, L¹⁰ is a bond.

In accordance with the foregoing referenced formulas, in someembodiments of a compound of Formula (V) or (VI), L¹¹ is substituted orunsubstituted —(CH₂CH₂O)_(n)—, substituted or unsubstituted—(OCH₂CH₂)_(n)—, substituted or unsubstituted —(CH₂)_(n)—, or bond. Insome embodiments, L¹¹ is substituted or unsubstituted —(CH₂CH₂O)_(n)—.In some embodiments, L¹¹ is substituted or unsubstituted—(OCH₂CH₂)_(n)—. In some embodiments, L¹¹ is substituted orunsubstituted —(CH₂)_(n)—. In some embodiments, L¹¹ is a bond. In someembodiments, n is 30 to 50. In some embodiments, n is 30 to 40. In someembodiments, n is 40 to 50.

In accordance with the foregoing referenced formulas, in someembodiments of a compound of Formula (V) or (VI), L¹² is substituted orunsubstituted C₁-C₁₂ alkylene, substituted or unsubstituted C₁-C₁₂heteroalkylene, substituted or unsubstituted C₂-C₁₂ alkenylene,substituted or unsubstituted C₂-C₁₂ alkynylene, —(CH₂CH₂O)_(m)—,—(OCH₂CH₂)_(m)—, —O—, —S—, —S(═O)—, —S(═O)₂—, —S(═O)(═NR¹)—, —C(═O)—,—C(═N—OR¹)—, —C(═O)O—, —OC(═O)—, —C(═O)C(═O)—, —C(═O)NR¹—, —NR¹C(═O)—,—OC(═O)NR¹—, —NR¹C(═O)O—, —NR¹C(═O)NR¹—, —C(═O)NR¹C(═O)—, —S(═O)₂NR¹—,—NR¹S(═O)₂—, —NR¹—, —N(OR¹)—, —O[(P═O)O⁻]O—, —O[(P═O)S⁻]O—, or a bond.In some embodiments, L¹² is substituted or unsubstituted C₁-C₁₂alkylene. In some embodiments, L¹² is substituted or unsubstitutedC₁-C₁₂ heteroalkylene. In some embodiments, L¹² is substituted orunsubstituted C₂-C₁₂ alkenylene. In some embodiments, L¹² is substitutedor unsubstituted C₂-C₁₂ alkynylene. In some embodiments, L¹² is—(CH₂CH₂O)_(m)— or —(OCH₂CH₂)_(m)—. In some embodiments, L¹² is —O—. Insome embodiments, L¹² is —S—. In some embodiments, L¹² is —S(═O)—. Insome embodiments, L¹² is —S(═O)₂—. In some embodiments, L¹² is—S(═O)(═NR¹)—. In some embodiments, L¹² is —C(═O)—. In some embodiments,L¹² is —C(═N—OR¹)—. In some embodiments, L¹² is —C(═O)O—. In someembodiments, L¹² is OC(═O)—. In some embodiments, L¹² is —C(═O)C(═O)—.In some embodiments, L¹² is —C(═O)NR¹—. In some embodiments, L¹² is—NR¹C(═O)—. In some embodiments, L¹² is —OC(═O)NR¹—. In someembodiments, L¹² is —NR¹C(═O)O—. In some embodiments, L¹² is—NR¹C(═O)NR¹—. In some embodiments, L¹² is —C(═O)NR¹C(═O)—. In someembodiments, L¹² is —S(═O)₂NR¹—. In some embodiments, L¹² is—NR¹S(═O)₂—. In some embodiments, L¹² is —NR¹—. In some embodiments, L¹²is —N(OR¹)—. In some embodiments, L¹² is —O[(P═O)O⁻]O—. In someembodiments, L¹² is —O[(P═O)S⁻]O—. In some embodiments, L¹² issubstituted or unsubstituted C₁-C₆ alkylene. In some embodiments, L¹² issubstituted or unsubstituted C₁-C₃ alkylene. In some embodiments, L¹² issubstituted or unsubstituted C₂-C₃ alkylene. In some embodiments, L¹² is—CH₂CH₂—. In some embodiments of a compound of Formula (V) or (VI), L¹²is —NR¹C(═O)O—. In some embodiments, L¹² is a bond. In some embodiments,L¹² is an organic molecular residue that intercalates with group R. Insome embodiments, L¹² can ionically/electrostatically interact with abase pair or covalently bond with a base pair. Some non-limitingexamples of an organic molecular residue that intercalates with group Rcan include berberine, ethidium bromide, daunomycin, thalidomide,doxorubicin (adriamycin), aflatoxin B1, amsacrine, acridines (e.g.,proflavine, quinacrine, acridine orange, Pyrazoloacridine), acriflavin,amonafide, 1,10-phenanthroline, metal cations with polycyclic aromaticligands (e.g. metals such as Rh(III); ligands such as Ir(III),dipyridine, terpyridine), bleomycin, actinomycin D, and ellipticine.

In some embodiments of a compound of Formula (V) or (VI), m is aninteger selected from 1 to 10. In some embodiments, m is selected from 1to 3. In some embodiments, m is selected from 1 to 5. In someembodiments, m is selected from 3 to 8. In some embodiments, m isselected from 2 to 5. In some embodiments, m is selected from 5 to 10.In some embodiments, m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In someembodiments, m is 1. In some embodiments, m is 2. In some embodiments, mis 3.

In some embodiments of a compound of Formula (V) or (VI), n is aninteger selected from 1 to 200. In some embodiments, n is selected from1 to 20. In some embodiments, n is selected from 1 to 50. In someembodiments, n is selected from 1 to 100. In some embodiments, n isselected from 50 to 100. In some embodiments, n is selected from 25 to50. In some embodiments, n is selected from 30 to 40. In someembodiments, n is selected from 25 to 75. In some embodiments, n isselected from 100 to 200. In some embodiments, n is selected from 50 to150. In some embodiments, n is selected from 150 to 200.

In some embodiments of a compound of Formula (VI), Formula (VIa), orFormula (VIb), m is an integer selected from 1 to 10. In someembodiments, m is selected from 1 to 3. In some embodiments, m isselected from 1 to 5. In some embodiments, m is selected from 3 to 8. Insome embodiments, m is selected from 2 to 5. In some embodiments, m isselected from 5 to 10. In some embodiments, m is 1, 2, 3, 4, 5, 6, 7, 8,9 or 10. In some embodiments, m is 1. In some embodiments, m is 2. Insome embodiments, m is 3.

In some embodiments of a compound of Formula (VI), Formula (VIa), orFormula (VIb), n is an integer selected from 1 to 200. In someembodiments, n is selected from 1 to 20. In some embodiments, n isselected from 1 to 50. In some embodiments, n is selected from 1 to 100.In some embodiments, n is selected from 50 to 100. In some embodiments,n is selected from 25 to 50. In some embodiments, n is selected from 30to 40. In some embodiments, n is selected from 25 to 75. In someembodiments, n is selected from 100 to 200. In some embodiments, n isselected from 50 to 150. In some embodiments, n is selected from 150 to200.

In some embodiments of a compound of Formula (V), Formula (VI), Formula(VIa), or Formula (VIb), each R¹ is independently H or —CH₃. In someembodiments, R¹ is H.

In some embodiments of a compound of Formula (V), Formula (VI), Formula(VIa), or Formula (VIb), R comprises one or more of fatty alcohols,fatty acids, glycerolipids, glycerophospholipids, sphingolipids,saccharolipids, polyketides, sterol lipids, and prenol lipids. In someembodiments, the R comprises one or more fatty alcohols. In someembodiments, each fatty alcohol is independently a saturated,monounsaturated, or polyunsaturated fatty alcohol. In some embodiments,the fatty alcohol comprises one or more a C₂-C₂₆ fatty alcohol. In someembodiments, the fatty alcohol comprises two or more a C₂-C₂₆ fattyalcohol. In some embodiments, each fatty alcohol is a C12, C14, C16,C18, C20, or C22 fatty alcohol. In some embodiments, each fatty alcoholis independently docosahexaenol, eicosapentaenol, oleyl alcohol, stearylalcohol, (9Z,12Z)-octadeca-9,12-dien-1-yl alcohol, (Z)-docos-13-en-1-ylalcohol, docosanyl alcohol, (E)-octadec-9-en-1-yl alcohol, icosanylalcohol, (9Z,12Z,15Z)-octadeca-9,12,15-trien-1-yl alcohol, or palmitylalcohol. In some embodiments, each fatty alcohol is a stearyl alcohol.In some embodiments, the R comprises one or more sterol lipids. In someembodiments, the R comprises one or more of vitamins. In someembodiments, each vitamin is independently a vitamin A, vitamin D,vitamin E, or vitamin K.

In some embodiments, R group provided in Formula (V), Formula (VI),Formula (VIa), or Formula (VIb) comprises a payload as described herein.In some embodiments, R group provided in Formula (V), Formula (VI),Formula (VIa), or Formula (VIb) comprises a lipid.

In some embodiments, R group provided in Formula (V), Formula (VI),Formula (VIa), or Formula (VIb) comprises a nucleic acid. In someembodiments, the nucleic acid is a single-stranded nucleic acid. In someembodiments, single-stranded nucleic acid is a DNA. In some embodiments,single-stranded nucleic acid is an RNA. In some embodiments, the nucleicacid is a double-stranded nucleic acid. In some embodiments, thedouble-stranded nucleic acid is a DNA. In some embodiments, thedouble-stranded nucleic acid is an RNA. In some embodiments, thedouble-stranded nucleic acid is a DNA-RNA hybrid. In some embodiments,the nucleic acid is a messenger RNA (mRNA), a microRNA, an asymmetricalinterfering RNA (aiRNA), a small hairpin RNA (shRNA), or aDicer-Substrate dsRNA. In some embodiments, the nucleic acid is an mRNA.In some embodiments, R comprises an mRNA molecule encoding a Casnuclease, i.e., a Cas nuclease mRNA. In some embodiments, R comprisesone or more guide RNAs or nucleic acids encoding guide RNAs. In someembodiments, R comprises a template nucleic acid for repair orrecombination. In some embodiments, R comprises an mRNA encoding a geneeditor nuclease. In some embodiments, R comprises an mRNA encoding abase editor nuclease. In some embodiments, R comprises an mRNA encodinga restriction enzyme. In some embodiments, R comprises zinc-fingernuclease or TALEN nuclease. In some embodiments, R comprises a guideRNA. In some embodiments, the gRNA hybridizes a gene selected fromPCSK9, ANGPTL3, APOC3, LPA, APOB, MTP, ANGPTL4, ANGPTL8, APOA5, APOE,LDLR, IDOL, NPC1L1, ASGR1, TM6SF2, GALNT2, GCKR, LPL, MLXIPL, SORT1,TRIB1, MARC1, ABCG5, and ABCG8. In some embodiments, the gRNA hybridizeswith PCSK9. In some embodiments, the gRNA hybridizes with ANGPTL3. Insome embodiments, R comprises a guide RNA sequence as described herein.In some embodiments, R comprises a coupling sequence as describedherein. In some embodiments, R comprises an mRNA, guide RNA, siRNA,antisense oligonucleotides, microRNA, decoy RNA, or aptamer. In someembodiments, when R is an nucleic acid, L¹² can intercalate with or bindto group R.

In some embodiments, R group provided in Formula (V), Formula (VI),Formula (VIa), or Formula (VIb) comprises an amino acid. In someembodiment, the amino acid is a natural amino acid. In some embodiment.the amino acid is an amino acid that is outside the 20 canonical aminoacids. The amino acid can be modified.

In some embodiments, R group provided in Formula (V), Formula (VI),Formula (VIa), or Formula (VIb) comprises a protein. In someembodiments, the protein is an Argonaute protein. In some embodiments,the protein is a cas protein. In some embodiments, the protein is anRNP.

In some embodiments, R group provided in Formula (V), Formula (VI),Formula (VIa), or Formula (VIb) comprises a lipid nanoparticle.

It is to be understood that the linkage between L¹² and R can be acovalent bond, a hydrogen bond, intermolecular or intramolecularinteraction.

In some embodiments of a compound of Formula (V), Formula (VI), Formula(VIa), or Formula (VIb), A is

In some embodiments of a compound of Formula (V), Formula (VI), Formula(VIa), or Formula (VIb), A is galactose.

In some embodiments, receptor targeting conjugates described herein areGalNAc-conjugated lipids that have a structure given in Table 4.

TABLE 4 Exemplary GalNAc-conjugated lipids

1001

1002

1003

1004

1005

1006

1007

1008

1009

1010

1011

1012

1013

1014

1015

1016

1017

1018

1019

1020

1021

1022

1023

1024

1025

1026

1027

1028

1029

1030

1031

1032

1033

1034

1035

1036

1037 n = 1-60

1038

1039

1040

1041

1042

1043

1044

1045

1046

1047

1048

1049

1050

1051

1052

1053

1054

1055

1056

1057

1058

1059

1060

1061

1062

1063

1064

1065

1066

1067

1068

1069

1070

1071

1072

1073

1074

1075

1076

1077

1078

1079

1080

1081

1082

1083

1084

1085

Each asymmetric carbon in Table 4 represents racemic, R and Sconfiguration unless otherwise specified. As shown in Table 4, each ofn, p, and q is independently 0, or an integer from 1 to 200. In someembodiments, each of n, p, and q of Table 4 is independently 0, or aninteger from 1 to 100. In some embodiments, each of n, p, and q of Table4 is independently 0, or an integer from 1 to 50. In some embodiments,each of n, p, and q of Table 4 is independently 0, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 14, 13, 15, 16, 17, 18, 19, or 20. In someembodiments, each of n, p, and q of Table 4 is independently 0, 1, 2, 3,4, or 5. In some embodiments, each of n, p, and q of Table 4 isindependently 0, 1, 2, or 3. In some embodiments, each of n, p, and q ofTable 4 is independently 1 or 2. In some embodiments, n is 1-60 and eachof p and q is independently 1-9 in Table 4. In some implementations, theexemplary GalNAc-conjugated lipids ID numbers 1001, 1011, 1015, 1020,1025, 1030, 1037, 1040, 1041, 1045, 1050, 1055, 1060, 1061, 1062, 1063,1064, 1065, 1066, 1067, and 1082 from Table 4 have n=1, 11, 36, or 44.In some embodiments of the exemplary GalNAc-conjugated lipids of Table4, n is 1 to 100. In some embodiments, n is 1 to 50. In someembodiments, n is 25 to 50. In some embodiments, n is 1 to 10. In someembodiments, n is 1 to 5. In some embodiments, n is 1 to 50. In someembodiments, n is 25 to 75. In some embodiments, n is 100 to 150. Insome embodiments, n is 1. In some embodiments, n is 11. In someembodiments, n is 36. In some embodiments, n is 44. In some embodiments,n is 40 to 50. In some embodiments, n is 30 to 40. In some embodimentsof the exemplary GalNAc-conjugated lipids of Table 4, p is 1 to 100. Insome embodiments, p is 1 to 50. In some embodiments, p is 25 to 50. Insome embodiments, p is 1 to 10. In some embodiments, p is 1 to 5. Insome embodiments, p is 1 to 50. In some embodiments, p is 25 to 75. Insome embodiments, p is 100 to 150. In some embodiments, p is 40 to 50.In some embodiments, p is 30 to 40. In some embodiments of the exemplaryGalNAc-conjugated lipids of Table 4, q is 1 to 100. In some embodiments,q is 1 to 50. In some embodiments, q is 25 to 50. In some embodiments, qis 1 to 10. In some embodiments, q is 1 to 5. In some embodiments, q is1 to 50. In some embodiments, q is 25 to 75. In some embodiments, q is100 to 150. In some embodiments, q is 40 to 50. In some embodiments, qis 30 to 40.

Lipid Nanoparticle (LNP) Compositions

In one aspect, disclosed herein are lipid nanoparticle compositions thatcomprise a receptor targeting conjugate as described herein. In someembodiments, disclosed herein are lipid nanoparticle compositions thatcomprise (i) a payload, such as a therapeutic agent, or a target ofinterest and (ii) a receptor targeting conjugate as described herein. Insome embodiments, disclosed herein are lipid nanoparticle compositionsthat comprise (i) one or more nucleic acid molecular entities (i.e.,nucleic acids such as mRNA and gRNA) and (ii) a receptor targetingconjugate as described herein. In some embodiments, herein describednanoparticle compositions comprise two or more receptor targetingconjugates, which conjugates can be the same or different. In someembodiments, the one or more nucleic acid molecular entities comprise anucleic acid described herein. In some embodiments, the one or morenucleic acid molecular entities comprise a single guide RNA (sgRNA) orguide RNA (gRNA) targeting a disease causing gene of interest producedin the hepatocytes. In some embodiments, the one or more nucleic acidmolecular entities comprise an mRNA that encodes a Cas nuclease. In someembodiments, at least one of the one or more nucleic acid molecularentities comprises a chemical modification, e.g., a chemicalmodification as described herein. In some embodiments, the chemicalmodification is a 2′-F modification, a phosphorothioate internucleotidelinkage modification, acyclic nucleotides, LNA, HNA, CeNA,2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy,2′-fluoro, 2′-O—N-methylacetamido (2′-O-NMA), a2′-O-dimethylaminoethoxyethyl (2′-0-DMAEOE), 2′-0-aminopropyl (2′-O-AP),4′-O-methyl, or a 2′-ara-F modification. In some embodiments, thechemical modification is a 2′-O-methyl modification.

In some embodiments, the receptor targeting conjugate comprises fromabout 0.001 mol % to about 20 mol % of the total lipid content presentin a herein described nanoparticle composition. In some embodiments, thereceptor targeting conjugate comprises from about 0.01 mol % to about 1mol % of the total lipid content present in a herein describednanoparticle composition. In some embodiments, the receptor targetingconjugate comprises from about 0.001 mol %, about 0.005 mol %, about0.01 mol %, about 0.02 mol %, about 0.03 mol %, about 0.04 mol %, about0.05 mol %, about 0.06 mol %, about 0.07 mol %, about 0.08 mol %, orabout 0.09 mol %, to about 1 mol %, about 1.5 mol %, about 2 mol %,about 5 mol %, about 10 mol %, or about 20 mol % of the total lipidcontent present in a herein described nanoparticle composition. In someembodiments, the receptor targeting conjugate comprises from about 0.001mol %, about 0.005 mol %, about 0.01 mol %, about 0.02 mol %, about 0.03mol %, about 0.04 mol %, or about 0.05 mol %, to about 0.06 mol %, about0.07 mol %, about 0.08 mol %, about 0.09 mol %, about 1 mol %, about 1.5mol %, about 2 mol %, about 5 mol %, about 10 mol %, or about 20 mol %of the total lipid content present in a herein described nanoparticlecomposition. In some embodiments, the receptor targeting conjugatecomprises about 0.01 mol %, about 0.02 mol %, about 0.03 mol %, about0.04 mol %, about 0.05 mol %, about 0.06 mol %, about 0.07 mol %, about0.08 mol %, about 0.09 mol %, about 0.1 mol %, about 0.2 mol %, about0.3 mol %, about 0.4 mol %, about 0.5 mol %, about 0.6 mol %, about 0.7mol %, about 0.8 mol %, about 0.9 mol %, about 1 mol %, about 1.1 mol %,about 1.2 mol %, about 1.3 mol %, about 1.4 mol %, about 1.5 mol %,about 1.6 mol %, about 1.7 mol %, about 1.8 mol %, about 1.9 mol %,about 2.0 mol %, about 3.0 mol %, about 4.0 mol %, or about 5.0 mol % ofthe total lipid content present in a herein described nanoparticlecomposition.

In some embodiments, an LNP described herein comprises from about0.000001 mol % to about 30 mol % of the receptor targeting conjugatebased on total lipid or total excipient content. In some embodiments, anLNP described herein comprises from about 0.0001 mol % to about 25 mol %of the receptor targeting conjugate based on total lipid or totalexcipient content. In some embodiments, an LNP described hereincomprises from about 0.0001, 0.001, 0.005, 0.01, 0.025, 0.05, or 0.25mol % to about 0.5, 1, 1.125, 1.25, 1.5, 1.75, 2, 5, 10, 15, 20 or 25mol % of the receptor targeting conjugate based on total lipid or totalexcipient content. In some embodiments, an LNP described hereincomprises from about 0.001 mol % to about 1 mol % of the receptortargeting conjugate based on total lipid or total excipient content. Insome embodiments, an LNP described herein comprises from about 0.005 mol% to about 1 mol % of the receptor targeting conjugate based on totallipid or total excipient content. In some embodiments, an LNP describedherein comprises from about 0.025 mol % to about 1, 1.5 or 2 mol % ofthe receptor targeting conjugate based on total lipid or total excipientcontent. In some embodiments, an LNP described herein comprises fromabout 0.25 mol % to about 1 mol % of the receptor targeting conjugatebased on total lipid or total excipient content. In some embodiments, anLNP described herein comprises from about 0.25 mol % to about 1.5 or 2mol % of the receptor targeting conjugate based on total lipid or totalexcipient content. In some embodiments, an LNP described hereincomprises from about 0.05 mol % to about 1.5 or 2 mol % of the receptortargeting conjugate based on total lipid or total excipient content. Insome embodiments, an LNP described herein comprises from about 0.05 mol% to about 1 mol % of the receptor targeting conjugate based on totallipid or total excipient content. In some embodiments, an LNP describedherein comprises from about 0.001 mol % to about 2 mol % of the receptortargeting conjugate based on total lipid or total excipient content. Insome embodiments, an LNP described herein comprises from about 0.005 mol% to about 2 mol % of the receptor targeting conjugate based on totallipid or total excipient content. In some embodiments, an LNP describedherein comprises at least about 0.001, 0.005, 0.01, 0.05, 0.1, 0.25,0.75, or 1 mol % of the receptor targeting conjugate based on totallipid or total excipient content. In some embodiments, an LNP describedherein comprises at most about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mol% of the receptor targeting conjugate based on total lipid or totalexcipient content. In some embodiments, an LNP described hereincomprises at most about 1 mol % of the receptor targeting conjugatebased on total lipid or total excipient content. In some embodiments, anLNP described herein comprises at most about 2 mol % of the receptortargeting conjugate based on total lipid or total excipient content.

In some embodiments, the herein described LNP compositions are sized onthe order of micrometers or smaller and can include a lipid bilayer.Nanoparticle compositions encompass lipid nanoparticles (LNPs),liposomes (e.g., lipid vesicles), and lipoplexes. For example, ananoparticle composition may be a liposome having a lipid bilayer with adiameter of 500 nm or less. The LNPs described herein can have a meandiameter of from about 1 nm to about 2500 nm, from about 10 nm to about1500 nm, from about 20 nm to about 1000 nm, from about 30 nm to about150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm,from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, fromabout 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80nm to about 90 nm, or from about 70 nm to about 80 nm. The LNPsdescribed herein can have a mean diameter of about 30 nm, 35 nm, 40 nm,45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140nm, 145 nm, 150 nm, or greater. The LNPs described herein can besubstantially non-toxic.

Cholesterol

In some embodiments, a herein described LNP composition comprises acholesterol or a derivative thereof. In some embodiments, the LNPcomposition comprises a structural lipid. The structural lipid can beselected from steroid, sterol, alkyl resoreinol, cholesterol orderivative thereof, fecosterol, sitosterol, ergosterol, campesterol,stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid,alpha-tocopherol, and a combination thereof. In some embodiments, thestructural lipid is a corticosteroid such as prednisolone,dexamethasone, prednisone, and hydrocortisone. In some embodiments, thecholesterol or derivative thereof is cholesterol,5-heptadecylresorcinol, or cholesterol hemisuccinate. In someembodiments, the cholesterol or derivative thereof is cholesterol.

In some embodiments, the cholesterol or derivative thereof is acholesterol derivative. In some embodiments, the cholesterol derivativeis a polar cholesterol analogue. In some embodiments, the polarcholesterol analogue is 5α-cholestanol, 5β-coprostanol,cholesteryl-(2′-hydroxy)-ethyl ether, cholesteryl-(4′-hydroxy)-butylether, or 6-ketocholestanol. In some embodiments, the polar cholesterolanalogue is cholesteryl-(4′-hydroxy)-butyl ether. In some embodiments,the cholesterol derivative is a non-polar cholesterol analogue. In someembodiments, the non-polar cholesterol analogue is 5α-cholestane,cholestenone, 5α-cholestanone, 5β-cholestanone, or cholesteryldecanoate.

In some embodiments, the cholesterol or the derivative thereof comprisesfrom 20 mol % to 50 mol % of the total lipid present in the nanoparticlecomposition. In some embodiments, the cholesterol or the derivativethereof comprises about 20 mol %, about 21 mol %, about 22 mol %, about23 mol %, about 24 mol %, about 25 mol %, about 26 mol %, about 27 mol%, about 28 mol %, about 29 mol %, about 30 mol %, about 31 mol %, about32 mol %, about 33 mol %, about 34 mol %, about 35 mol %, about 36 mol%, about 37 mol %, about 38 mol %, about 39 mol %, about 40 mol %, about41 mol %, about 42 mol %, about 43 mol %, about 44 mol %, about 45 mol%, about 46 mol %, about 47 mol %, about 48 mol %, or about 50 mol % ofthe total lipid present in the nanoparticle composition.

Phospholipid

In some embodiments, a herein described LNP composition comprises aphospholipid. In some embodiments, the phospholipid comprises a lipidselected from the group consisting of: phosphatidylcholine (PC),phosphatidylethanolamine amine, glycerophospholipid,sphingophospholipids, Guriserohosuhono, sphingolipids phosphono lipids,natural lecithins, and hydrogenated phospholipid. In some embodiments,the phospholipid comprises a phosphatidylcholine. Exemplaryphosphatidylcholines include, but are not limited to, soybeanphosphatidylcholine, egg yolk phosphatidylcholine (EPC),distearoylphosphatidylcholine,1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), dipalmitoylphosphatidylcholine, dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),2-Oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC), dimyristoylphosphatidylcholine (DMPC), and dioleoyl phosphatidylcholine (DOPC). Incertain specific embodiments, the phospholipid is DSPC.

In some embodiments, the phospholipid comprises aphosphatidylethanolamine amine. In some embodiments, thephosphatidylethanolamine amine is distearoyl phosphatidylethanolamine(DSPE), dipalmitoyl phosphatidyl ethanolamine (DPPE),1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), dimyristoylphosphoethanolamine (DMPE), 16-0-Monome Le PE, 16-0-dimethyl PE,18-1-trans PE, palmitoyl oleoyl-phosphatidylethanolamine (POPE), or1-stearoyl-2-oleoyl-phosphatidyl ethanolamine (SOPE). In someembodiments, the phospholipid comprises a glycerophospholipid. In someembodiments, the glycerophospholipid is plasmalogen, phosphatidate, orphosphatidylcholine. In some embodiments, the glycerophospholipid isphosphatidylserine, phosphatidic acid, phosphatidylglycerol,phosphatidylinositol, palmitoyl oleoyl phosphatidylglycerol (POPG), orlysophosphatidylcholine. In some embodiments, the phospholipid comprisesa sphingophospholipid. In some embodiments, the sphingophospholipid issphingomyelin, ceramide phosphoethanolamine, ceramide phosphoglycerol,or ceramide phosphoglycerophosphoric acid. In some embodiments, thephospholipid comprises a natural lecithin. In some embodiments, thenatural lecithin is egg yolk lecithin or soybean lecithin. In someembodiments, the phospholipid comprises a hydrogenated phospholipid. Insome embodiments, the hydrogenated phospholipid is hydrogenated soybeanphosphatidylcholine. In some embodiments, the phospholipid is selectedfrom the group consisting of: phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,phosphatidic acid, palmitoyloleoyl phosphatidylcholine,lysophosphatidylcholine, lysophosphatidylethanolamine,dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine,distearoylphosphatidylcholine, and dilinoleoylphosphatidylcholine.

In some embodiments, the phospholipid comprises a lipid selected from:1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC),1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC),2-Oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC),1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC),1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine(OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC),1,2-dilinolenoyl-sn-glycero-3-phosphocholine,1,2-diarachidonoyl-sn-glycero-3-phosphocholine,1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine,1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE),1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine,1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine,1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine,1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine,1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG),and sphingomyelin.

A phospholipid can comprise a phospholipid moiety and one or more fattyacid moieties. A phospholipid moiety can comprise phosphatidyl choline,phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine,phosphatidic acid, 2-lysophosphatidyl choline, or a sphingomyelin. Afatty acid moiety can comprise lauric acid, myristic acid, myristoleicacid, palmitic acid, palmitoleic acid, stearic acid, oleic acid,linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid,arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid,docosapentaenoic acid, or docosahexaenoic acid. In some specificembodiments, a phospholipid can be functionalized with or cross-linkedto one or more alkynes, which may undergo a copper-catalyzedcycloaddition upon exposure to an azide.

In some embodiments, the LNP composition comprises a plurality ofphospholipids, for example, at least 2, 3, 4, 5, or more distinctphospholipids. In some embodiments, the phospholipid comprises from 1mol % to 20 mol % of the total lipid present in the LNP composition. Insome embodiments, the phospholipid comprises from about 5 mol % to about15 mol % of the total lipid present in the LNP composition. In someembodiments, the phospholipid comprises from about 8 mol % to about 12mol % of the total lipid present in the LNP composition. In someembodiments, the phospholipid comprises from about 5 mol %, about 6 mol%, about 7 mol %, about 8 mol %, about 9 mol %, about 10 mol %, about 11mol %, about 12 mol %, about 13 mol %, about 14 mol %, or about 15 mol %of the total lipid present in the LNP composition. In some embodiments,the phospholipid comprises from about 9 mol %, about 10 mol %, or about11 mol % of the total lipid present in the LNP composition.

Stealth Lipids

In some embodiments, a herein described LNP composition comprises astealth lipid. “Stealth lipids” can refer to lipids that alter thelength of time the nanoparticles can exist in vivo (e.g., in the blood).Stealth lipids can assist in the formulation process by, for example,reducing particle aggregation and controlling particle size. Stealthlipids used herein may modulate pharmacokinetic properties of the LNP.Stealth lipids suitable for use in a lipid composition of the disclosurecan include, but are not limited to, stealth lipids having a hydrophilichead group linked to a lipid moiety. Stealth lipids suitable for use ina lipid composition of the present disclosure and information about thebiochemistry of such lipids can be found in Romberg et al,Pharmaceutical Research, Vol. 25, No. 1, 2008, pg. 55-71 and Hoekstra etal, Biochimica et Biophysica Acta 1660 (2004) 41-52. Additional suitablePEG lipids are disclosed, e.g., in WO 2006/007712.

In some embodiments, the stealth lipid is a PEG-lipid. In oneembodiment, the hydrophilic head group of stealth lipid comprises apolymer moiety selected from polymers based on PEG (sometimes referredto as poly(ethylene oxide)), poly(oxazoline), poly(vinyl alcohol),poly(glycerol), poly(N-vinylpyrrolidone), polyaminoacids and polyN-(2-hydroxypropyl)methacrylamide]. Stealth lipids can comprise a lipidmoiety. In some embodiments, the lipid moiety of the stealth lipid maybe derived from diacylglycerol or diacylglycamide, including thosecomprising a dialkylglycerol or dialkylglycamide group having alkylchain length independently comprising from about C4 to about C40saturated or unsaturated carbon atoms, wherein the chain may compriseone or more functional groups such as, for example, an amide or ester.The dialkylglycerol or dialkylglycamide group can further comprise oneor more substituted alkyl groups.

PEG-Lipid

In some embodiments, a described LNP composition comprises a PEG-lipid.In some embodiments, the described LNP composition comprises two or morePEG-lipids. Exemplary PEG-lipids include, but are not limited to, thelipids in Table 2. Exemplary PEG-lipids also include, but are notlimited to, PEG-modified phosphatidylethanolamines, PEG-modifiedphosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines,PEG-modified diacylglycerols, PEG-modified dialkylglycerols, andmixtures thereof. For example, the one or more PEG-lipids can comprisePEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, a PEG-DSPE lipid, ora combination thereof. In some embodiments, PEG moiety is an optionallysubstituted linear or branched polymer of ethylene glycol or ethyleneoxide. In some embodiments, the PEG moiety is substituted, e.g., by oneor more alkyl, alkoxy, acyl, hydroxy, or aryl groups. In someembodiments, the PEG moiety includes PEG copolymer such asPEG-polyurethane or PEG-polypropylene (see, e.g., j. Milton Harris,Poly(ethylene glycol) chemistry: biotechnical and biomedicalapplications (1992)). In some embodiments, the PEG moiety does notinclude PEG copolymers, e.g., it may be a PEG monopolymer. ExemplaryPEG-lipids include, but are not limited to, PEG-dilauroylglycerol,PEG-dimyristoylglycerol (PEG-DMG), PEG-dipalmitoylglycerol,PEG-distearoylgiycerol (PEG-DSPE), PEG-dipalmitoylglycerol,PEG-disterylglycerol, PEG-dilaurylglycamide, PEG-dimyristylglycamide,PEG-dipalmitoylglycamide, PEG-disterylglycamide, PEG-cholesterol, andPEG-DMB (3,4-Ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol)ether),1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000]).

In some embodiments, a PEG-lipid is a PEG-lipid conjugate, for example,PEG coupled to dialkyloxypropyls (e.g., PEG-DAA conjugates), PEG coupledto diacylglycerols (e.g., PEG-DAG conjugates), PEG coupled tocholesterol, PEG coupled to phosphatidylethanolamines, and PEGconjugated to ceramides (see, e.g., U.S. Pat. No. 5,885,613), cationicPEG lipids, polyoxazoline (POZ)-lipid conjugates (e.g., POZ-DAAconjugates; see, e.g., WO 2010/006282), polyamide oligomers (e.g.,ATTA-lipid conjugates), and mixtures thereof.

A PEG-lipid can comprise one or more ethylene glycol units, for example,at least 1, at least 2, at least 5, at least 10, at least 20, at least30, at least 40, at least 50, at least 60, at least 70, at least 80, atleast 90, at least 100, at least 120, or at least 150 ethylene glycolunits. In some embodiments, a number average molecular weight of thePEG-lipids is from about 200 Da to about 5000 Da. In some embodiments, anumber average molecular weight of the PEG-lipids is from about 500 Dato about 3000 Da. In some embodiments, a number average molecular weightof the PEG-lipids is from about 750 Da to about 2500 Da. In someembodiments, a number average molecular weight of the PEG-lipids is fromabout 750 Da to about 2500 Da. In some embodiments, a number averagemolecular weight of the PEG-lipids is about 500 Da, about 750 Da, about1000 Da, about 1250 Da, about 1500 Da, about 1750 Da, or about 2000 Da.In some embodiments, a polydispersity index (PDI) of the one or morePEG-lipids is smaller than 2. In some embodiments, a PDI of the one ormore PEG-lipids is at most 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0. In someembodiments, a PDI of the one or more PEG-lipids is at least 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,2.7, 2.8, 2.9, or 3.0.

In some embodiments, the PEG-lipid comprises from about 0.1 mol % toabout 10 mol % of the total lipid present in the LNP composition. Insome embodiments, the PEG-lipid comprises from about 0.1 mol % to about6 mol % of the total lipid present in the LNP composition. In someembodiments, the PEG-lipid comprises from about 0.5 mol % to about 5 mol% of the total lipid present in the LNP composition. In someembodiments, the PEG-lipid comprises from about 1 mol % to about 3 mol %of the total lipid present in the LNP composition. In some embodiments,the PEG-lipid comprises about 2.0 mol % to about 2.5 mol % of the totallipid present in the LNP composition. In some embodiments, the PEG-lipidcomprises about 1 mol %, about 1.1 mol %, about 1.2 mol %, about 1.3 mol%, about 1.4 mol %, about 1.5 mol %, about 1.6 mol %, about 1.7 mol %,about 1.8 mol %, about 1.9 mol %, about 2.0 mol %, about 2.1 mol %,about 2.2 mol %, about 2.3 mol %, about 2.4 mol %, about 2.5 mol %,about 2.6 mol %, about 2.7 mol %, about 2.8 mol %, about 2.9 mol %, orabout 3.0 mol % of the total lipid present in the LNP composition.

Amino Lipid

In some embodiments, an LNP composition described herein comprises anamino lipid. In some embodiments, the LNP comprises a plurality of aminolipids. For example, the LNP composition can comprise 2, 3, 4, 5, 6, 7,8, 9, 10, or more amino lipids. For another example, the LNP compositioncan comprise at least 2, at least 3, at least 4, at least 5, at least 6,at least 7, at least 9, at least 10, or at least 20 amino lipids. Foryet another example, the LNP composition can comprise at most 2, at most3, at most 4, at most 5, at most 6, at most 7, at most 9, at most 10, atmost 20, or at most 30 amino lipids.

In some embodiments, an LNP composition described herein comprises oneor more amino lipids. In some embodiments, the one or more amino lipidscomprise from about 1 mol % to about 65 mol % of the total lipid presentin the LNP composition. In some embodiments, the one or more aminolipids comprise from about 10 mol % to about 60 mol % of the total lipidpresent in the LNP composition. In some embodiments, the one or moreamino lipids comprise from about 40 mol % to about 65 mol % of the totallipid present in the LNP composition. In some embodiments, the one ormore amino lipids comprise from about 10 mol %, about 15 mol %, about 20mol %, about 25 mol %, about 30 mol %, about 35 mol % or about 40 mol %to about 45 mol %, 50 mol %, 55 mol %, 60 mol %, or about 65 mol % ofthe total lipid present in the LNP composition. In some embodiments, theone or more amino lipids comprise about 40 mol %, about 41 mol %, about42 mol %, about 43 mol %, about 44 mol %, about 45 mol %, about 46 mol%, about 47 mol %, about 48 mol %, about 49 mol %, about 50 mol %, about51 mol %, about 52 mol %, about 53 mol %, about 54 mol %, about 55 mol%, about 56 mol %, about 57 mol %, about 58 mol %, about 59 mol %, about60 mol %, about 61 mol %, about 62 mol %, about 63 mol %, about 64 mol%, or about 65 mol % of the total lipid present in the LNP composition.In some embodiments, the LNP composition comprises a first amino lipidand a second amino lipid. In some embodiments, the first amino lipidcomprises from about 1 mol % to about 99 mol % of the total amino lipidspresent in the LNP composition. In some embodiments, the first aminolipid comprises from about 16.7 mol % to about 66.7 mol % of the totalamino lipids present in the LNP composition. In some embodiments, thefirst amino lipid comprises from about 20 mol % to about 60 mol % of thetotal amino lipids present in the LNP composition.

In some embodiments, the amino lipid is an ionizable lipid. An ionizablelipid can comprise one or more ionizable nitrogen atoms. In someembodiments, at least one of the one or more ionizable nitrogen atoms ispositively charged. In some embodiments, at least 10 mol %, 20 mol %, 30mol %, 40 mol %, 50 mol %, 60 mol %, 70 mol %, 80 mol %, 90 mol %, 95mol %, or 99 mol % of the ionizable nitrogen atoms in the LNPcomposition are positively charged. In some embodiments, the amino lipidcomprises a primary amine, a secondary amine, a tertiary amine, animine, an amide, a guanidine moiety, a histidine residue, a lysineresidue, an arginine residue, or any combination thereof. In someembodiments, the amino lipid comprises a primary amine, a secondaryamine, a tertiary amine, a guanidine moiety, or any combination thereof.In some embodiments, the amino lipid comprises a tertiary amine.

In some embodiments, the amino lipid is a cationic lipid. In someembodiments, the amino lipid is an ionizable lipid. In some embodiments,the amino lipid comprises one or more nitrogen atoms. In someembodiments, the amino lipid comprises one or more ionizable nitrogenatoms. Exemplary cationic and/or ionizable lipids include, but are notlimited to, 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine(KL10),N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine(KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25),1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA),2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA),heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate(DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)[1,3]-dioxolane(DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA),2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA),(2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA (2R)), and(2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA (2S)). Further examples of the amino lipids suitable forthe present disclosure can be found in US 20180290965A1, WO2017/173054A1, US 20150273068A1, WO 2015/095340A1, U.S. Pat. Nos.9,365,610, 8,193,246, 8,192,753, 9,549,983, 8,017,804, 8,357,722,7,799,565, and 7,745,651, all of which are hereby incorporated byreference in their entirety.

In some embodiments, an amino lipid described herein can take the formof a salt, such as a pharmaceutically acceptable salt. Allpharmaceutically acceptable salts of the amino lipid are encompassed bythis disclosure. As used herein, the term “amino lipid” also includesits pharmaceutically acceptable salts, and its diastereomeric,enantiomeric, and epimeric forms.

In some embodiments, an amino lipid described herein, possesses one ormore stereocenters and each stereocenter exists independently in eitherthe R or S configuration. The lipids presented herein include alldiastereomeric, enantiomeric, and epimeric forms as well as theappropriate mixtures thereof. The lipids provided herein include allcis, trans, syn, anti, entgegen (E), and zusammen (Z) isomers as well asthe appropriate mixtures thereof.

Payload

In one aspect, the herein described LNP compositions comprise a payload.The LNP compositions described herein can be designed to deliver apayload, such as a therapeutic agent, or a target of interest. Exemplarytherapeutic agents include, but are not limited to, antibodies (e.g.,monoclonal, chimeric, humanized, nanobodies, and fragments thereofetc.), cholesterol, hormones, peptides, proteins, chemotherapeutics andother types of antineoplastic agents, low molecular weight drugs,vitamins, co-factors, nucleosides, nucleotides, oligonucleotides,enzymatic nucleic acids, antisense nucleic acids, triplex formingoligonucleotides, antisense DNA or RNA compositions, chimeric DNA:RNAcompositions, allozymes, aptamers, ribozyme, decoys and analogs thereof,plasmids and other types of expression vectors, and small nucleic acidmolecules, RNAi agents, short interfering nucleic acid (siNA), messengerribonucleic acid (messenger RNA, mRNA), short interfering RNA (siRNA),double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA(shRNA) molecules, peptide nucleic acid (PNA), a locked nucleic acidribonucleotide (LNA), morpholino nucleotide, threose nucleic acid (TNA),glycol nucleic acid (GNA), sisiRNA (small internally segmentedinterfering RNA), aiRNA (asymmetrical interfering RNA), and siRNA with1, 2 or more mismatches between the sense and anti-sense strand torelevant cells and/or tissues, such as in a cell culture, subject ororganism. Therapeutic agents can be purified or partially purified, andcan be naturally occurring or synthetic, or chemically modified. In someembodiments, the therapeutic agent is an RNAi agent, short interferingnucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA(dsRNA), micro-RNA (miRNA), or a short hairpin RNA (shRNA) molecule. Insome embodiments, the therapeutic agent is an mRNA.

In some embodiments, the payload comprises one or more nucleic acid(s)(i.e., one or more nucleic acid molecular entities). In someembodiments, the nucleic acid is a single-stranded nucleic acid. In someembodiments, single-stranded nucleic acid is a DNA. In some embodiments,single-stranded nucleic acid is an RNA. In some embodiments, the nucleicacid is a double-stranded nucleic acid. In some embodiments, thedouble-stranded nucleic acid is a DNA. In some embodiments, thedouble-stranded nucleic acid is an RNA. In some embodiments, thedouble-stranded nucleic acid is a DNA-RNA hybrid. In some embodiments,the nucleic acid is a messenger RNA (mRNA), a microRNA, an asymmetricalinterfering RNA (aiRNA), a small hairpin RNA (shRNA), or aDicer-Substrate dsRNA.

In some embodiments, the payload comprises an mRNA. In some embodiments,the payload comprises an mRNA molecule encoding a Cas nuclease, i.e., aCas nuclease mRNA. In some embodiments, the payload comprises one ormore guide RNAs or nucleic acids encoding guide RNAs. In someembodiments, the payload comprises a template nucleic acid for repair orrecombination. In some embodiments, the payload comprises an mRNAencoding a gene editor nuclease. In some embodiments, the payloadcomprises an mRNA encoding a base editor nuclease. In some embodiments,the payload comprises an mRNA encoding a restriction enzyme. In someembodiments, the payload comprises zinc-finger nuclease or TALENnuclease.

In some embodiments, the mRNA payload, such as a Cas nuclease mRNA, canbe modified for improved stability and/or immunogenicity properties. Themodifications may be made to one or more nucleosides within the mRNA.Examples of chemical modifications to mRNA nucleobases includepseudouridine, 1-methyl-pseudouridine, and 5-methyl-cytidine. Additionalmodifications to improve stability, expression, and immunogenicity canalso be made. The mRNA encoding a Cas nuclease can be codon optimizedfor expression in a particular cell type, such as a eukaryotic cell, amammalian cell, or more specifically, a human cell. In some embodiments,the mRNA encodes a human codon optimized Cas9 nuclease or human codonoptimized Cpf nuclease as the Cas nuclease. In some embodiments, themRNA encodes a gene editor (i.e., genome editor) nuclease and is calleda gene editor mRNA. In some embodiments, the gene editor is a Casprotein, such as the ones described herein. In some embodiments, thegene editor is an engineered nuclease. In some embodiments, the geneeditor introduces a double stranded break in a gene of interest. In someembodiments, the gene editor introduces a double stranded break at atargeted point within a gene of interest. In some embodiments, the geneeditor introduces a single stranded break in a gene of interest. In someembodiments, the gene editor is a base editor. In some embodiments, thegene editor inserts a nucleic acid sequence into a gene of interest. Insome embodiments, the gene editor deletes a targeted sequence from agene of interest. In some embodiments, the gene editor mRNA encodes Cas9nuclease. In some embodiments, the gene editor mRNA encodes base editornuclease. In some embodiments, the gene editor mRNA encodes arestriction enzyme. In some embodiments, the gene editor mRNA encodeszinc-finger nuclease. In some embodiments, the gene editor mRNA encodestranscription activator-like effector-based nucleases (TALEN). In someembodiments, the gene editor mRNA encodes a meganuclease. In someembodiments, the gene editor mRNA encodes an Argonaute protein. In someembodiments, the mRNA is purified. In some embodiments, the mRNA ispurified using a precipitation method (e.g., LiCl precipitation, alcoholprecipitation, or an equivalent method, e.g., as described herein) or achromatography-based method (e.g., an HPLC-based method or an equivalentmethod).

In some embodiments, the Cas nuclease mRNA comprises a 3′ or 5′untranslated region (UTR). In some embodiments, the 3′ or 5′ UTR can bederived from a human gene sequence. Exemplary 3′ and 5′ UTRs include α-and β-globin, albumin, HSD17B4, and eukaryotic elongation factor 1a. Inaddition, viral-derived 5′ and 3′ UTRs can also be used and includeorthopoxvirus and cytomegalovirus UTR sequences. In certain embodiments,the mRNA includes a 5′ cap, such as m7G(5′)ppp(5′)N. In certainembodiments, this cap can be a cap-0 where nucleotide N does not contain2′OMe, or cap-1 where nucleotide N contains 2′OMe, or cap-2 wherenucleotides N and N+1 contain 2′OMe. In some embodiments, the 5′ cap canregulate nuclear export; prevent degradation by exonucleases; promotetranslation; and promote 5′ proximal intron excision. In addition, capscan also contain a non-nucleic acid entity that acts as the bindingelement for eukaryotic translation initiation factor 4E, eIF4E. Incertain embodiments, the mRNA includes a poly(A) tail. This tail can beabout 40 to about 300 nucleotides in length. In some embodiments, thetail is about 40 to about 100 nucleotides in length. In someembodiments, the tail is about 100 to about 300 nucleotides in length.In some embodiments, the tail is about 100 to about 300 nucleotides inlength. In some embodiments, the tail is about 50 to about 200nucleotides in length. In some embodiments, the tail is about 50 toabout 250 nucleotides in length. In certain embodiments, the tail isabout 100, 150, or 200 nucleotides in length. The poly(A) tail cancontain modifications to prevent exonuclease degradation includingphosphorothioate linkages and modifications to the nucleobase. In someembodiments, the poly(A) tail contains a 3′ “cap” which could includemodified or non-natural nucleobases or other synthetic moieties. In someembodiments, the mRNA comprises at least one element that is capable ofmodifying the intracellular half-life of the RNA. The half-life of theRNA can be increased or decreased. In some embodiments, the element iscapable of increasing or decreasing the stability of the RNA. In someembodiments the element may promote RNA decay. In some embodiments, theelement can activate translation. In some embodiments, the element maybe within the 3′ UTR of the RNA. For example, the element may be an mRNAdecay signal or may include a polyadenylation signal (PA).

In some embodiments, the Cas nuclease mRNA encodes a Cas protein from aCRISPR/Cas system. In some embodiments, the Cas protein comprises atleast one domain that interacts with a guide RNA (“gRNA”). In someembodiments, the Cas protein is directed to a target sequence by a guideRNA. The guide RNA can interact with the Cas protein as well as thetarget sequence such that, it can direct binding to the target sequence.In some embodiments, the guide RNA provides the specificity for thetargeted cleavage, and the Cas protein may be universal and paired withdifferent guide RNAs to cleave different target sequences. In certainembodiments, the Cas protein may cleave single or double-stranded DNA.In certain embodiments, the Cas protein may cleave RNA. In certainembodiments, the Cas protein may nick RNA. In some embodiments, the Casprotein comprises at least one DNA binding domain and at least onenuclease domain. In some embodiments, the nuclease domain may beheterologous to the DNA binding domain. In certain embodiments, the Casprotein may be modified to reduce or eliminate nuclease activity. TheCas protein may be used to bind to and modulate the expression oractivity of a DNA sequence.

In some embodiments, the CRISPR/Cas system comprises Class 1 or Class 2system components, including ribonucleic acid protein complexes. TheClass 2 Cas nuclease families of proteins are enzymes with DNAendonuclease activity, and they can be directed to cleave a desirednucleic acid target by designing an appropriate guide RNA, as describedfurther herein. A Class 2 CRISPR/Cas system component may be from aType-IIA, Type-IIB, Type-IIC, Type V, or Type VI system. Class 2 Casnucleases include, for example, Cas9, Cpf1, C2c1, C2c2, and C2c3proteins. In some embodiments, the Cas protein is from a Type-IICRISPR/Cas system, i.e., a Cas9 protein from a CRISPR/Cas9 system, or aType-V CRISPR/Cas system, e.g., a Cpf1 protein. In some embodiments, theCas protein is from a Class 2 CRISPR/Cas system, i.e., a single-proteinCas nuclease such as a Cas9 protein or a Cpf1 protein.

Exemplary species that the Cas9 protein or other components can be frominclude, but are not limited to, Streptococcus pyogenes, Streptococcusthermophilus, Streptococcus sp., Staphylococcus aureus, Listeriainnocua, Lactobacillus gasseri, Francisella novicida, Wolinellasuccinogenes, Sutterella wadsworthensis, Gamma proteobacterium,Neisseria meningitidis, Campylobacter jejuni, Pasteurella multocida,Fibrobacter succinogene, Rhodospirillum rubrum, Nocardiopsisdassonvillei, Streptomyces pristinaespiralis, Streptomycesviridochromogenes, Streptomyces viridochromogenes, Streptosporangiumroseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides,Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillusdelbrueckii, Lactobacillus salivarius, Lactobacillus buchneri, Treponemadenticola, Microscilla marina, Burkholderiales bacterium, Polar omonasnaphthalenivorans, Polar omonas sp., Crocosphaera watsonii, Cyanothecesp., Microcystis aeruginosa, Synechococcus sp., Acetohalobiumarabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, CandidatusDesulforudis, Clostridium botulinum, Clostridium difficile, Finegoldiamagna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum,Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatiumvinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcuswatsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer,Methanohalobium evestigatum, Anabaena variabilis, Nodular ia spumigena,Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp.,Lyngbya sp., Microcoleus chthonoplastes, Oscillator ia sp., Petrotogamobilis, Thermosipho africanus, Streptococcus pasteurianus, Neisseriacinerea, Campylobacter lari, Parvibaculum lavamentivorans, Corynebacterium diphtheria, or Acaryochloris marina. In some embodiments, theCas9 protein is from Streptococcus pyogenes. In some embodiments, theCas9 protein may be from Streptococcus thermophilus. In someembodiments, the Cas9 protein is from Staphylococcus aureus.

In some embodiments, the payload comprises at least one guide RNA. Theguide RNA may guide the Class 2 Cas nuclease to a target sequence on atarget nucleic acid molecule, where the guide RNA hybridizes with andthe Cas nuclease cleaves or modulates the target sequence. In someembodiments, a guide RNA binds with and provides specificity of cleavageby a Class 2 nuclease. In some embodiments, the guide RNA and the Casprotein may form a ribonucleoprotein (RNP), e.g., a CRISPR/Cas complex.In some embodiments, the CRISPR complex may be a Type-II CRISPR/Cas9complex. In some embodiments, the CRISPR/Cas complex may be a Type-VCRISPR/Cas complex, such as a Cpf1/guide RNA complex. In someembodiments, the Cas nuclease may be a single-protein Cas nuclease, e.g.a Cas9 protein or a Cpf 1 protein. In some embodiments, the guide RNAtargets cleavage by a Cas9 protein. In some embodiments, the payloadcomprises two or more guide RNA molecules. In some embodiments, the twoor more guide RNA molecules target the same disease-causing gene. Insome embodiments, the two or more guide RNA molecules target differentgenes. In some specific embodiments, the two guide RNA molecules targettwo separate disease-causing genes of interest.

A guide RNA for a CRISPR/Cas9 nuclease system comprises a CRISPR RNA(crRNA) and a tracr RNA (tracr). In some embodiments, the crRNA maycomprise a targeting sequence that is complementary to and hybridizeswith the target sequence on the target nucleic acid molecule. The crRNAmay also comprise a flagpole that is complementary to and hybridizeswith a portion of the tracrRNA. In some embodiments, the crRNA mayparallel the structure of a naturally occurring crRNA transcribed from aCRISPR locus of a bacteria, where the targeting sequence acts as thespacer of the CRISPR/Cas9 system, and the flagpole corresponds to aportion of a repeat sequence flanking the spacers on the CRISPR locus.The guide RNA may target any sequence of interest via the targetingsequence of the crRNA. In some embodiments, the degree ofcomplementarity between the targeting sequence of the guide RNA and thetarget sequence on the target nucleic acid molecule is at least about60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In someembodiments, the targeting sequence of the guide RNA and the targetsequence on the target nucleic acid molecule may be 100% complementary.In other embodiments, the targeting sequence of the guide RNA and thetarget sequence on the target nucleic acid molecule may contain at leastone mismatch. For example, the targeting sequence of the guide RNA andthe target sequence on the target nucleic acid molecule may contain 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches. In some embodiments, thetargeting sequence of the guide RNA and the target sequence on thetarget nucleic acid molecule may contain 1-6 mismatches.

In some embodiments, the length of the targeting sequence depends on theCRISPR/Cas system and components used. For example, different Casproteins from different bacterial species have varying optimal targetingsequence lengths. Accordingly, the targeting sequence may comprise 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 35, 40, 45, 50, or more than 50 nucleotides inlength. In some embodiments, the targeting sequence comprised 18-24nucleotides in length. In some embodiments, the targeting sequencecomprises 19-21 nucleotides in length. In some embodiments, thetargeting sequence comprises 20 nucleotides in length.

In some embodiments, the guide RNA is a “dual guide RNA” or “dgRNA”. Insome embodiments, the dgRNA comprises a first RNA molecule comprising acrRNA, and a second RNA molecule comprising a tracr RNA. The first andsecond RNA molecules may form a RNA duplex via the base pairing betweenthe flagpole on the crRNA and the tracr RNA. In some embodiments, theguide RNA is a “single guide RNA” or “sgRNA”. In some embodiments, thesgRNA may comprise a crRNA covalently linked to a tracr RNA. In someembodiments, the crRNA and the tracr RNA may be covalently linked via alinker. In some embodiments, the single-molecule guide RNA may comprisea stem-loop structure via the base pairing between the flagpole on thecrRNA and the tracr RNA. In some embodiments, the sgRNA is a “Cas9sgRNA” capable of mediating RNA-guided DNA cleavage by a Cas9 protein.In certain embodiments, the guide RNA comprises a crRNA and tracr RNAsufficient for forming an active complex with a Cas9 protein andmediating RNA-guided DNA cleavage. In some embodiments, the payloadcomprises more than one guide RNAs; each guide RNA contains a differenttargeting sequence, such that the CRISPR/Cas system cleaves more thanone target sequence. In some embodiments, one or more guide RNAs mayhave the same or differing properties such as activity or stabilitywithin a CRISPR/Cas complex. Where more than one guide RNA is used, eachguide RNA can be encoded on the same or on different expressioncassettes. The promoters used to drive expression of the more than oneguide RNA may be the same or different.

In some embodiments, the nucleic acid payload, such as RNAs, ismodified. Modified nucleosides or nucleotides can be present in a guideRNA or mRNA. A guide RNA or Cas nuclease encoding mRNA comprising one ormore modified nucleosides or nucleotides is called a “modified” RNA todescribe the presence of one or more non-naturally and/or naturallyoccurring components or configurations that are used instead of or inaddition to the canonical A, G, C, and U residues. In some embodiments,a modified RNA is synthesized with a non-canonical nucleoside ornucleotide. Modified nucleosides and nucleotides can include one or moreof: (i) alteration, e.g., replacement, of one or both of the non-linkingphosphate oxygens and/or of one or more of the linking phosphate oxygensin the phosphodiester backbone linkage (an exemplary backbonemodification); (ii) alteration, e.g., replacement, of a constituent ofthe ribose sugar, e.g., of the 2′ hydroxyl on the ribose sugar (anexemplary sugar modification); (iii) wholesale replacement of thephosphate moiety with “dephospho” linkers (an exemplary backbonemodification); (iv) modification or replacement of a naturally occurringnucleobase, including with a non-canonical nucleobase (an exemplary basemodification); (v) replacement or modification of the ribose-phosphatebackbone (an exemplary backbone modification); (vi) modification of the3′ end or 5′ end of the oligonucleotide, e.g., removal, modification orreplacement of a terminal phosphate group or conjugation of a moiety,cap or linker (such 3′ or 5′ cap modifications may comprise a sugarand/or backbone modification); and (vii) modification or replacement ofthe sugar (an exemplary sugar modification).

In some embodiments, the payload can include a template nucleic acid.The template can be used to alter or insert a nucleic acid sequence ator near a target site for a Cas nuclease. In some embodiments, thetemplate is used in homologous recombination. In some embodiments, thehomologous recombination may result in the integration of the templatesequence or a portion of the template sequence into the target nucleicacid molecule. In some embodiments, a single template is provided. Inother embodiments, two or more templates are provided such thathomologous recombination may occur at two or more target sites.

In some embodiments, the payload, such as one or more RNAs, are fullyencapsulated within the lipid portion of the particle, therebyprotecting the RNAs from nuclease degradation. Fully encapsulated canindicate that the RNA in the nucleic acid-lipid particle is notsignificantly degraded after exposure to serum or a nuclease assay thatwould significantly degrade free DNA or RNA. In some embodiments, thenucleic acid-lipid particle composition comprises a RNA molecule that isfully encapsulated within the lipid portion of the particles, such thatfrom about 30% to about 100%, from about 40% to about 100%, from about50% to about 100%, from about 60% to about 100%, from about 70% to about100%, from about 80% to about 100%, from about 90% to about 100%, fromabout 30% to about 95%, from about 40% to about 95%, from about 50% toabout 95%, from about 60% to about 95%, from about 70% to about 95%,from about 80% to about 95%, from about 85% to about 95%, from about 90%to about 95%, from about 30% to about 90%, from about 40% to about 90%,from about 50% to about 90%, from about 60% to about 90%, from about 70%to about 90%, from about 80% to about 90%, or at least about 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% (or any fraction thereof or rangetherein) of the particles have the RNA encapsulated therein.

In some embodiments, the payload comprises an mRNA and one or more guideRNA. In some embodiments, the mRNA encodes a gene editor nuclease and iscalled a gene editor mRNA. In some embodiments, the gene editor mRNAencodes Cas9 nuclease. In some embodiments, the mRNA encodes base editornuclease. In some embodiments, the gene editor mRNA encodes zinc-fingernuclease. In some embodiments, the gene editor mRNA encodes TALENnuclease.

Additional Compositions of the LNP

In some embodiments, an LNP composition described herein comprises oneor more antioxidants. In some embodiments, the one or more antioxidantsfunction to reduce a degradation of the cationic lipids, the payload, orboth. In some embodiments, the one or more antioxidants comprise ahydrophilic antioxidant. In some embodiments, the one or moreantioxidants is a chelating agent such as ethylenediaminetetraaceticacid (EDTA) and citrate. In some embodiments, the one or moreantioxidants is EDTA. In some embodiments, the one or more antioxidantscomprise a lipophilic antioxidant. In some embodiments, the lipophilicantioxidant comprises a vitamin E isomer or a polyphenol. In someembodiments, the one or more antioxidants are present in the LNPcomposition at a concentration of at least 1 mM, at least 10 mM, atleast 20 mM, at least 50 mM, or at least 100 mM. In some embodiments,the one or more antioxidants are present LNP composition at aconcentration of about 20 mM.

Method of Making Lipid Nanoparticles

Described in the present disclosure are innovative processes for makingLNP compositions, e.g., LNPs comprising a receptor targeting conjugatesuch as a GalNAc-lipid.

Traditionally, LNPs comprising GalNAc-lipids are prepared by apost-addition process (i.e., Post-addition of GalNAc-lipid), whichinvolves the addition of GalNAc-lipids into pre-formed LNPs after anincubation period, followed by buffer exchange. The traditionally usedpost-addition process is illustrated as Process 1 in FIG. 9.

While the traditional process of post-addition of receptor targetingconjugates such as GalNAc-lipids into pre-formed LNPs is effective inpreparing nanoparticles, the innovative processes described herein(e.g., by adding GalNAc-Lipid into LNP excipients, by split addition, orby successively introducing GalNAc-lipid through a third channel/portinto the inline mixing chamber) offer significant advantages over thepost-addition or post-insertion of GalNAc-lipid. The said advantagesinclude, but are not limited to, more homogenous distribution ofGalNAc-lipid across lipid nanoparticles and better process control overpost-insertion and downstream processing of GalNAc-LNPs. For example, insome cases, the lipid nanoparticles prepared by a process involving theaddition of GalNAc-lipid into LNP excipients have better particleuniformity and/or provide better editing efficacy than correspondinglipid nanoparticles prepared by post-addition of GalNAc-lipid. In somecases, the lipid nanoparticles prepared by a process involving splitaddition of GalNAc-lipid have better particle uniformity and/or providebetter editing efficacy than corresponding lipid nanoparticles preparedby post-addition of GalNAc-lipid. Similarly, the successive third portinsertion of GalNAc-lipid into the inline mixing chamber produced betterparticle uniformity and/or better editing efficacy than correspondingcorresponding lipid nanoparticles prepared by post-insertion ofGalNAc-lipid.

Provided in the present disclosure is a method of making a formulationcomprising the herein-described nanoparticles. In some embodiments, thelipid nanoparticles comprise (i) one or more nucleic acid molecularentities, (ii) one or more lipids selected from a sterol or a derivativethereof, a phospholipid, a stealth lipid, and an amino lipid, and/or(iii) a receptor targeting conjugate. Accordingly, in one aspect,described herein is a method of making a formulation comprising lipidnanoparticles that comprise (i) one or more nucleic acid molecularentities, (ii) one or more lipids selected from a sterol or a derivativethereof, a phospholipid, a stealth lipid, and an amino lipid, and (iii)a receptor targeting conjugate. In some embodiments, the receptortargeting conjugate is a GalNAc-lipid. In some embodiments, theGalNAc-lipid is selected from a compound of Table 4. In someembodiments, the GalNAc-lipid is compound 1004 in Table 4. In someembodiments, the GalNAc-lipid is compound 1053, 1014, 1043, 1002, 1044,1004 in Table 4, or a combination thereof. In some embodiments, receptortargeting conjugate is a compound in Table 4. In some embodiments, thereceptor targeting conjugate comprises one or more N-acetylgalactosamine(GalNAc) or GalNAc derivatives. In some embodiments, the receptortargeting conjugate comprises a structure of Formula (I), Formula (Ia),Formula (Ib), Formula (II), Formula (IIa), Formula (IIb), Formula (IIc),Formula (III), Formula (IIIa), Formula (IIIb), Formula (IIIc), Formula(IIId), Formula (IIIe), Formula (IV), Formula (V), Formula (VI), Formula(VIa), or Formula (VIb).

A process for making lipid nanoparticles can comprise several generalsteps: (i) providing an aqueous solution, such as citrate or phosphatebuffer, comprising one or more nucleic acid molecular entities in afirst reservoir; (ii) providing a second solution comprising one or morelipids and an organic solvent, such as an alcohol (e.g., ethanol) in asecond reservoir; and (iii) mixing the aqueous solution with the secondsolution. The first reservoir is optionally in fluid communication withthe second reservoir.

The process can optionally comprise one or more dilution steps, one ormore incubation steps, one or more buffer exchange steps, one or moreconcentration steps, and/or one or more filtrations steps. In someembodiments, the dilution step involves dilution by adding a dilutionbuffer. In some embodiments, the dilution step involves dilution withaqueous buffer (e.g. citrate buffer or pure water) e.g., using a pumpingapparatus (e.g. a peristaltic pump). In some embodiments, the dilutionbuffer is an organic solution such as alcohol. The dilution step cancomprise a dilution that is 1 to 20 times of the initial volume, or anynumbers or ranges therebetween. In some embodiments, the dilution stepcomprises a dilution that is 1 to 10 times of the initial volume. Insome embodiments, the dilution step is followed by the buffer exchangestep or the incubation step. In some embodiments, the dilution buffercomprises one or more lipids, such as a sterol or a derivative thereof,a phospholipid, a stealth lipid, an amino lipid, a GalNAc-lipid, or acombination thereof. In some embodiments, the dilution buffer comprisesstealth lipid. In some embodiments, the stealth lipid is present in thedilution buffer at 0.01 mol % to 5 mol %. In some embodiments, thedilution buffer comprises GalNAc-lipid. In some embodiments, theGalNAc-lipid is present in the dilution buffer at 0.01 mol % to 10 mol%, or any numbers or ranges therebetween. In some embodiments, a portionof the GalNAc-lipid present in the nanoparticles is introduced throughthe dilution buffer.

The incubation step comprises allowing a solution from the mixing stepto stand in a vessel for about 0 to about 100 hours at about roomtemperature and optionally protected from light. In some embodiments,the incubation step runs from 0 to 24 hours, 1 minute to 2 hours, or 1minute to 60 minutes. In some embodiments, the incubation step runs from1 minutes to 120 minutes. In some embodiments, the incubation step isfollowed by the buffer exchange step. In some embodiments, theincubation step follows the buffer exchange step.

In some embodiments, the buffer exchange step comprises a solventexchange that results in a higher concentration of phosphate bufferedsaline (PBS) buffer. In some embodiments, the buffer exchange stepcomprises removing all or a portion of organic solvent. In someembodiments, the buffer exchange step comprises dialysis through asuitable membrane (e.g. 10,000 mwc snakeskin membrane). In someembodiments, the buffer exchange step comprises filtration such astangential flow filtration (TFF)). In some embodiments, the bufferexchange step comprises chromatography such as using a desalting column,e.g., PD10 column. In some embodiments, the buffer exchange stepcomprises ultrafiltration. Ultrafiltration comprises concentration ofthe diluted solution followed by diafiltration, e.g., using a suitablepumping system (e.g. pumping apparatus such as a peristaltic pump orequivalent thereof) in conjunction with a suitable ultrafiltrationmembrane (e.g. GE Hollow fiber cartridges or equivalent).

In some embodiments, the mixing step provides a clear single phase. Insome embodiments, after the mixing step, the organic solvent is removedto provide a suspension of particles, wherein the one or more nucleicacid molecular entities are encapsulated by the lipid(s). The selectionof an organic solvent will typically involve consideration of solventpolarity and the ease with which the solvent can be removed at the laterstages of particle formation. The organic solvent, which can serve as asolubilizing agent, can be in an amount sufficient to provide a clearsingle phase mixture of the one or more nucleic acid molecular entitiesand lipid(s). The organic solvent may be selected from one or more(e.g., two) of chloroform, dichloromethane, diethylether, cyclohexane,cyclopentane, benzene, toluene, methanol, and other aliphatic alcohols(e.g. C₁ to C₈) such as ethanol, propanol, isopropanol, butanol,tert-butanol, iso-butanol, pentanol and hexanol. The methods used toremove the organic solvent can involve diafiltration or dialysis orevaporation at reduced pressures or blowing a stream of inert gas (e.g.nitrogen or argon) across the mixture.

In other embodiments, the method further comprises adding nonlipidpolycations which are useful to effect the transformation of cells usingthe present compositions. Examples of suitable nonlipid polycationsinclude, but are limited to, hexadimethrine bromide (sold under thebrand name POLYBRENE®, from Aldrich Chemical Co., Milwaukee, Wis., USA)or other salts of hexadimethrine. Other suitable polycations include,e.g., salts of poly-L-ornithine, poly-L-arginine, poly-L-lysine,poly-D-lysine, polyallylamine and polyethyleneimine. In certainembodiments, the formation of the lipid nanoparticles can be carried outeither in a mono-phase system (e.g. a Bligh and Dyer monophase orsimilar mixture of aqueous and organic solvents) or in a two-phasesystem with suitable mixing.

The lipid nanoparticles can be formed in a mono- or a bi-phase system.In some embodiments, in a mono-phase system, the amino lipid(s) and oneor more nucleic acid molecular entities are each dissolved in a volumeof the mono-phase mixture. Combining the two solutions provides a singlemixture in which the complexes form. In some embodiments, in a bi-phasesystem, the amino lipids bind to the one or more nucleic acid molecularentities (which is present in the aqueous phase), and thus increasingthe solubility in organic phase.

In some embodiments, the solution of sterol(s) or derivative(s) thereof,phospholipid lipid(s) and amino lipid(s) is a solution comprisingorganic solvent. In some embodiments, the solution of GalNAc-lipid(s)comprises organic solvent such as ethanol. In some embodiments, thestealth lipid is prepared in an aqueous solution. In some embodiments,the stealth lipid is prepared in an organic solution. Contacting the oneor more nucleic acid molecular entities with the organic solutioncomprising one or more lipids can be accomplished by mixing together afirst solution of the one or more nucleic acid molecular entities and asecond solution of the lipids.

In some embodiments, the lipid nanoparticles are prepared in anapparatus comprising a first reservoir for holding an aqueous solutionand a second reservoir for holding an organic lipid solution. In someembodiments, the apparatus comprises additional reservoirs for holdingan aqueous solution (such as for a portion of the one or more nucleicacid molecular entities) and/or an organic solution (such as for all ora portion of the GalNAc-lipid). The apparatus can include a pumpmechanism configured to pump the aqueous and the organic lipid solutionsinto a mixing region or mixing chamber at substantially equal flowrates. In some embodiments, the mixing region or mixing chambercomprises a T coupling or equivalent thereof, which allows the aqueousand organic fluid streams to combine as input into the T connector andthe resulting combined aqueous and organic solutions to exit out of theT connector into a collection reservoir or equivalent thereof.

In one aspect, described herein is a method of preparing a formulationcomprising nanoparticles, wherein the nanoparticles comprise (i) one ormore nucleic acid molecular entities, (ii) one or more lipids selectedfrom a sterol or a derivative thereof, a phospholipid, a stealth lipid,and an amino lipid, and (iii) a receptor targeting conjugate. In someembodiments, the method comprises (a) providing a first solutioncomprising at least one of the one or more nucleic acid molecularentities; (b) providing a second solution comprising at least one of theone or more lipids; (c) mixing the first solution and the secondsolution, thereby producing a mixture comprising nanoparticles thatcomprise the one or more nucleic acid molecular entities and the one ormore lipids; (d) combining the receptor targeting conjugate with the oneor more lipids; (e) optionally carrying out a incubating step; and (f)optionally carrying out a buffer exchange step. In some embodiments, themethod comprises (a) providing a first solution comprising the one ormore nucleic acid molecular entities; (b) providing a second solutioncomprising at least one of the one or more lipids; (c) mixing the firstsolution and the second solution, thereby producing a mixture comprisingnanoparticles that comprise the one or more nucleic acid molecularentities and the one or more lipids; (d) combining the receptortargeting conjugate with the one or more lipids; (e) incubating thenanoparticles; and (f) optionally carrying out a buffer exchange step.In some embodiments, the method comprises providing (a) a first solutioncomprising the one or more nucleic acid molecular entities; (b)providing a second solution comprising at least one of the one or morelipids; (c) mixing the first solution and the second solution, therebyproducing a mixture comprising nanoparticles that comprise the one ormore nucleic acid molecular entities and the one or more lipids; (d)combining the receptor targeting conjugate with the one or more lipids,wherein at least a portion of the receptor targeting conjugate iscombined with the one or more lipids prior to or concurrently with themixing step; (e) optionally incubating the nanoparticles; and (f)optionally carrying out a buffer exchange step.

In some embodiments, the receptor targeting conjugate is combined withthe one or more lipids after the mixing step. In some embodiments, thereceptor targeting conjugate is combined with the one or more lipidsprior the mixing step. In some embodiments, the receptor targetingconjugate is combined with the one or more lipids concurrently with themixing step. In some embodiments, at least a portion of the receptortargeting conjugate is combined with the one or more lipids concurrentlywith the mixing step. In some embodiments, at least a portion of thereceptor targeting conjugate is combined with the one or more lipidsprior to the mixing step. In some embodiments, the receptor targetingconjugate is combined with the one or more lipids in the secondsolution. In some embodiments, the receptor targeting conjugate iscombined with other components of the lipid nanoparticles afterincubating step. In some embodiments, the receptor targeting conjugateis combined with other components of the lipid nanoparticles after aconcentrating step. In some embodiments, the receptor targetingconjugate is combined with other components of the lipid nanoparticlesafter freeze-thawing the nanoparticles.

A receptor targeting conjugate described herein can be partially orfully combined with other components of the lipid nanoparticles afterthe one or more nucleic acid molecular entities are mixed with the oneor more lipids that are selected from a sterol or a derivative thereof,a phospholipid, a stealth lipid, and an amino lipid. FIGS. 10-11illustrate 6 exemplary protocols (protocols 1-6). In some embodiments,the receptor targeting conjugate is introduced after nucleic acidmolecular entities are mixed with a sterol or a derivative thereof, aphospholipid, a stealth lipid, and/or an amino lipid. In someembodiments, the receptor targeting conjugate is added in a dilutionbuffer. The dilution buffer can be mixed with preformed nucleicacid-lipid nanoparticles coming out of an inline mixing chamber therebyforming the nanoparticles. In some embodiments, the dilution buffercomprises at least a portion of the lipids such as stealth lipid. Insome embodiments, all the receptor targeting conjugate in an LNPcomposition are introduced in a dilution buffer. In some embodiments,the receptor targeting conjugate is introduced to the lipidnanoparticles after an addition of a dilution buffer to the mixture andholding the diluted mixture for a period of time. In some embodiments,the holding time is between 1 and 120 minutes. In some embodiments, theholding time is between 1 and 90 minutes, between 1 and 60 minutes, orbetween 10 and 40 minutes. In some embodiments, the holding time is fromabout 25 to 35 minutes, from about 20 to 40 minutes, from about 10 to 50minutes, or from about 5 to 60 minutes. In some embodiments, the holdingtime is about 10 minutes, about 15 minutes, about 20 minutes, about 25minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45minutes, or about 50 minutes. In some embodiments, the holding time isabout 30 minutes. In some embodiments, the receptor targeting conjugateis introduced to the nanoparticles after buffer exchange. In someembodiments, the receptor targeting conjugate is introduced to thenanoparticles immediately after buffer exchange. In some embodiments,the receptor targeting conjugate is introduced to the nanoparticlesafter buffer exchange and concentration, but prior to storage. In someembodiments, the receptor targeting conjugate is introduced to thenanoparticles after buffer exchange, but prior to concentration andstorage. In some embodiments, the receptor targeting conjugate isintroduced to the nanoparticles after storage and thawing, and prior todosing or evaluation.

A receptor targeting conjugate described herein can be partially orfully pre-mixed with the one or more lipids that are selected from asterol or a derivative thereof, a phospholipid, a stealth lipid, and anamino lipid, thereby being introduced to the nanoparticlessimultaneously with other components of the premix (i.e., addition ofGalNAc-lipid into LNP excipients). FIGS. 12-13 illustrate 5 exemplaryprotocols (protocols 7-11) of GalNAc-lipid addition into LNP excipients.Further exemplary protocol of addition of GalNAc-lipid into LNPexcipients are illustrated as Process 4 in FIG. 9.

A receptor targeting conjugate described herein can be partially orfully combined with other components of the lipid nanoparticles byinline mixing. For example, after the one or more nucleic acid molecularentities are mixed with the one or more lipids that are selected from asterol or a derivative thereof, a phospholipid, a stealth lipid, and anamino lipid, the receptor targeting conjugate can be successively addedvia inline mixing through a third channel or port. The successive inlinemixing can provide instantaneous mixing of the receptor targetingconjugate with the rest of components in the nanoparticles and therebyforming the target nanoparticles. In some embodiments, all or a portionof the receptor targeting conjugate is combined with other componentsvia cross-mixing. In some embodiments, all or a portion of the receptortargeting conjugate is combined with other components via a T-shapemixer. In some embodiments, all or a portion of the receptor targetingconjugate is combined with other components via a microfluidics mixer.FIG. 14 illustrates 2 exemplary protocols (protocols 12-13) ofGalNAc-lipid addition via inline mixing. Further exemplary protocol isillustrated as Process 3 in FIG. 9.

A receptor targeting conjugate described herein can be combined withother components of the lipid nanoparticles through two or moreseparate, independent additions (i.e., split addition of GalNAc-lipid).In some embodiments, the two or more separate additions are carried outat different steps. In some embodiments, the two or more separateadditions are carried out concurrently. In some embodiments, the two ormore separate additions involves different solutions. In someembodiments, a portion of the receptor targeting conjugate is combinedwith the one or more lipids in the second solution and a portion of thereceptor targeting conjugate is combined with the one or more lipidsafter the mixing. In some embodiments, a portion of the receptortargeting conjugate is combined with the one or more lipids in thesecond solution and a portion of the receptor targeting conjugate iscombined with the one or more lipids after the incubating step. In someembodiments, a portion of the receptor targeting conjugate is combinedwith the one or more lipids in the second solution and a portion of thereceptor targeting conjugate is combined with the one or more lipidsafter the buffer exchange step. FIGS. 12-13 illustrate 5 exemplaryprotocols (protocols 7-11) of split addition of GalNAc-lipid. Furtherexemplary protocols of split addition of GalNAc-lipid are illustrated inFIG. 14. Similarly, other components of the LNPs can be introduced bysplit addition. For example, as illustrated in Protocol 13 of FIG. 14,the one or more nucleic acid molecular entities can be introduced in twoseparate buffer solutions. In some embodiments, the one or more nucleicacid molecular entities are introduced in 2 to 4 solutions. In someembodiments, the sterol or a derivative thereof, the phospholipid, thestealth lipid, and/or the amino lipid are independently introduced tothe LNPs in 1-3 solutions, which can occur concurrently or at differenttimes.

In some embodiments, a method of making a formulation comprising theherein-described nanoparticles comprises diluting the mixture producedby mixing the first and the second solutions by adding a dilutionbuffer. In some embodiments, the mixture is diluted inline. In someembodiments, the dilution buffer comprises at least a portion of thereceptor targeting conjugate. In some embodiments, the dilution buffercomprises at least a portion of the stealth lipid.

In some embodiments, the first solution comprises an aqueous buffer. Insome embodiments, the first solution comprises an organic solvent. Insome embodiments, the first solution comprises a mixture of an aqueousbuffer mixed with an organic solvent. In some embodiments, the organicsolvent present in the aqueous buffer is ethanol. In some embodiments,the ethanol percentage in the aqeuous buffer ranges from 0.1% to 50%, orany numbers or ranges therebetween. In some embodiments, the secondsolution comprises a mixture of an aqueous buffer mixed with an organicsolvent. In some embodiments, the second solution comprises ethanol. Insome embodiments, the second solution comprises ethanol and water. Insome embodiments, the dilution buffer comprises an aqueous buffer. Insome embodiments, the dilution buffer comprises an organic solvent. Insome embodiments, the dilution buffer comprises ethanol and water. Insome embodiments, the dilution buffer comprises 10% to 20% of ethanol inPBS buffer.

In some embodiments, a receptor targeting conjugate such as GalNAc-lipidis introduced to the nanoparticles as a solution. In some embodiments,the concentration of the receptor targeting conjugate in the solution isfrom about 0.1 mol % to 20 mol %, or any numbers or ranges therebetween.In some embodiments, the concentration of the receptor targetingconjugate in the solution is from about 10 mol % to about 20 mol %, fromabout 5 mol % to about 10 mol %, from about 0.25 mol % to about 5 mol %,from about 0.5 mol % to about 3 mol %, from about 0.5 mol % to about 2mol %, from about 0.25 mol % to about 1 mol %, from about 0.25 mol % toabout 0.5 mol %, from about 1 mol % to about 2 mol %, from about 2 mol %to about 3 mol %, or from about 0.1 mol % to about 0.5 mol %. In someembodiments, the concentration of the receptor targeting conjugate inthe solution is about 0.25 mol %, about 0.5 mol %, about 0.9 mol %,about 1 mol %, about 1.5 mol %, or about 2 mol %. In some embodiments,the concentration of the receptor targeting conjugate in the solution isabout 0.25 mol %.

In some embodiments, the mixing comprises laminar mixing, vortex mixing,turbulent mixing, or a combination thereof. In some embodiments, themixing comprises cross-mixing. In some embodiments, the mixing comprisesinline mixing. In some embodiments, the mixing comprises introducing atleast a portion of the first solution through a first inlet channel andat least a portion of the second solution through a second inletchannel, and wherein an angle between the first inlet channel and thesecond inlet channel is from about 0 to 180 degrees. In someembodiments, the angle between the first inlet channel and the secondinlet channel is from about 15 to 180 degrees, from about 30 to 180degrees, from about 45 to 180 degrees, from about 60 to 180 degrees,from about 90 to 180 degrees, or any numbers or ranges therebetween. Insome embodiments, the mixing comprises introducing a portion of thefirst solution through a third inlet channel. The mixing step can takeplace by any number of methods, e.g., by mechanical means such as avortex mixer. In some embodiments, the mixing step comprises inlinemixing. Exemplary mixing processes are illustrated in FIGS. 9-14. Insome embodiments, the mixing step comprises cross-mixing GalNAc-lipid,as illustrated in FIG. 14. In some embodiments, the solution containingthe targeting conjugate is introduced into the inline mixing chamberthrough a third inlet.

In some embodiments, a method of making a formulation comprising theherein-described nanoparticles comprises a filtration step. In someembodiments, a method of making a formulation comprising theherein-described nanoparticles comprises buffer exchange. In someembodiments, the buffer exchange comprises dialysis, chromatography, ortangential flow filtration (TFF).

Pharmaceutical Composition

In one aspect, disclosed herein are pharmaceutical compositionscomprising one or more described LNP compositions. For example, apharmaceutical composition can include one or more LNP compositionsincluding one or more different payloads. In some embodiments, thepharmaceutical composition comprises two or more LNP compositions, whichcan be the same or different.

In one aspect, disclosed herein are pharmaceutical compositionscomprising one or more described receptor targeting conjugates. In someembodiments, the pharmaceutical composition comprises two or morereceptor targeting conjugates, which can be the same or different. Insome embodiments, disclosed herein are pharmaceutical compositions thatcomprise (i) a first receptor targeting conjugate or a firstnanoparticle composition, and (ii) a second receptor targeting conjugateor a second nanoparticle composition.

Pharmaceutical compositions can further include one or morepharmaceutically acceptable excipients, carrier, or accessoryingredients such as those described herein. General guidelines for theformulation and manufacture of pharmaceutical compositions and agentsare available, for example, in Remington's The Science and Practice ofPharmacy, 21st Edition, A. R. Gennaro; Lippincott, Williams & Wilkins,Baltimore, Md., 2006. Excipients or carriers can include any ingredientother than the compound(s) of the disclosure, the other lipidcomponent(s) and the payload. An excipient may impart either afunctional (e.g. drug release rate controlling) and/or a nonfunctional(e.g. processing aid or diluent) characteristic to the formulations. Thechoice of excipient and carrier can depend on factors such as theparticular mode of administration, the effect of the excipient onsolubility and stability, and the nature of the dosage form. Parenteralformulations are typically aqueous or oily solutions or suspensions.Excipients or carrier such as sugars (including but not restricted toglucose, mannitol, sorbitol, etc.), salts, carbohydrates and bufferingagents (preferably to a pH of from 3 to 9) can be used. In someembodiments, the LNP compositions can be formulated with a sterilenon-aqueous solution or as a dried form to be used in conjunction with asuitable vehicle such as sterile, pyrogen-free water (WFI).

In some embodiments, the excipient or carrier can make up greater than50% of the total mass or volume of a pharmaceutical compositioncomprising a nanoparticle composition. For example, the excipient orcarrier can make up 50%, 60%, 70%, 80%, 90%, or more of a pharmaceuticalcomposition. In some embodiments, a pharmaceutically acceptableexcipient or carrier is at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% pure. In some embodiments, apharmaceutical composition can comprise between 0.1% and 100% (wt/wt) ofone or more nanoparticle compositions. In certain embodiments, thenanoparticle compositions and/or pharmaceutical compositions arerefrigerated or frozen for storage and/or shipment (e.g., being storedat a temperature of 4° C. or lower, such as a temperature between about−150° C. and about 0° C. or between about −80° C. and about −20° C. Insome embodiments, the nanoparticle compositions and/or pharmaceuticalcompositions are refrigerated or frozen at about −5° C., −10° C., −15°C., −20° C., −25° C., −30° C., −40° C., −50° C., −60° C., −70° C., −80°C., −90° C., −130° C., or −150° C.

The described LNP compositions and/or pharmaceutical compositions can beadministered to any patient or subject, including those patients orsubjects that may benefit from a therapeutic effect provided by thedelivery of the payload to one or more particular cells, tissues,organs, or systems or groups thereof. In some embodiments, the subjectis a mammal such as human. In some embodiments, the subject is non-humanprimates or mammals, including commercially relevant mammals such ascattle, pigs, hoses, sheep, cats, dogs, mice, and/or rats.

A pharmaceutical composition including one or more nanoparticlecompositions can be prepared by any method known or hereafter developedin the art of pharmacology. In general, such preparatory methods includebringing the active ingredient into association with an excipient and/orone or more other accessory ingredients, and then, if desirable ornecessary, dividing, shaping, and/or packaging the product into adesired single- or multi-dose unit.

A pharmaceutical composition in accordance with the present disclosurecan be prepared, packaged, and/or sold in bulk, as a single unit dose,and/or as a plurality of single unit doses. As used herein, a “unitdose” is discrete amount of the pharmaceutical composition comprising apredetermined amount of the active ingredient (e.g., nanoparticlecomposition). The amount of the active ingredient is generally equal tothe dosage of the active ingredient which would be administered to asubject and/or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage. Pharmaceuticalcompositions may be prepared in a variety of forms suitable for avariety of routes and methods of administration. For example,pharmaceutical compositions may be prepared in liquid dosage forms(e.g., emulsions, microemulsions, nanoemulsions, solutions, suspensions,syrups, and elixirs), injectable forms, solid dosage forms (e.g.,capsules, tablets, pills, powders, and granules), dosage forms fortopical and/or transdermal administration (e.g., ointments, pastes,creams, lotions, gels, powders, solutions, sprays, inhalants, andpatches), suspensions, powders, and other forms.

In some embodiments, the pharmaceutical composition comprises 1, 2, 3,4, 5, 6, 7, 8, 9, 10 or more payloads. In some embodiments, thepharmaceutical composition comprises two distinct payloads, such guideRNA and mRNA. The guide RNA and mRNA can be located in the same LNPcomposition, or they can be located at separate LNP compositions. Forexample, a pharmaceutical composition can comprise two distinct LNPcompositions, one comprising a guide RNA payload and the othercomprising an mRNA payload. For another example, a pharmaceuticalcomposition can comprise two distinct LNP compositions, one comprising aguide RNA (or mRNA) payload and the other comprising both an mRNApayload and a guide RNA payload. For yet another example, apharmaceutical composition can comprise one LNP composition, whichcomprising an mRNA payload and a guide RNA payload. In some embodiments,the pharmaceutical composition comprises two or more distinct LNPcompositions. In some embodiments, the two or more distinct LNPcompositions are present in the pharmaceutical composition such that themRNA molecule(s) and the guide RNA molecule(s) are at a mole or weightratio described herein.

The gRNA and mRNA payloads can be present in the pharmaceuticalcomposition at various molar or weight ratios. For example, the gRNA tomRNA ratio in the pharmaceutical composition can be from 0.01 to 100 byweight, and/or any value therebetween. For example, the gRNA to mRNAratio in the pharmaceutical composition can be from 0.01 to 100 by mole,and/or any value therebetween. In some embodiments, the ratio of gRNA tomRNA in the pharmaceutical composition is from about 1 to about 50 byweight or by mole, and/or any value therebetween. In some embodiments,the ratio of gRNA to mRNA in the pharmaceutical composition is fromabout 0.1 to about 10 by weight or by mole, and/or any valuetherebetween. In some embodiments, the ratio of gRNA to mRNA in thepharmaceutical composition is from about 0.2 to about 5, from about 0.25to about 4, from about 0.3 to about 3, or from about 0.5 to about 2 byweight. In some embodiments, the ratio of gRNA to mRNA in thepharmaceutical composition is from about 0.2 to about 5, from about 0.25to about 4, from about 0.3 to about 3, or from about 0.5 to about 2 bymole. In some embodiments, the gRNA to mRNA ratio in the pharmaceuticalcomposition is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5,about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10 by weight. Insome embodiments, the gRNA to mRNA ratio in the pharmaceuticalcomposition is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5,about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10 by mole. Insome embodiments, the mRNA to gRNA ratio in the pharmaceuticalcomposition is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5,about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10 by weight. Insome embodiments, the mRNA to gRNA ratio in the pharmaceuticalcomposition is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5,about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10 by mole. Insome embodiments, the gRNA to mRNA ratio in the pharmaceuticalcomposition is about 1:1 by weight. In some embodiments, the gRNA tomRNA ratio in the pharmaceutical composition is about 1:1 by mole.

In some embodiments, the gRNA in the pharmaceutical composition targetsa disease-causing gene that is produced in the hepatocytes. In someembodiments, the pharmaceutical composition comprises more than oneguide RNA. For example, the pharmaceutical composition can comprise 2,3, 4, 5, or more distinct guide RNAs. In some embodiments, thepharmaceutical composition comprises two guide RNA molecules. In someembodiments, the pharmaceutical composition comprises one mRNA and twoor more guide RNA molecules. In some embodiments, the two or more guideRNA molecules target the same disease-causing gene. In some embodiments,the two or more guide RNA molecules target different genes. In somespecific embodiments, the two guide RNA molecules target two separatedisease-causing genes of interest produced in the hepatocytes. In someembodiments, the gRNA is a sgRNA. In some embodiments, the gRNA is adgRNA.

The LNP compositions and pharmaceutical compositions disclosed hereincan be used in methods for gene editing, both in vivo and in vitro. Insome embodiments, the methods comprise contacting a cell with an LNPcomposition or a pharmaceutical composition described herein. In someembodiments, the cell is a mammalian cell. In some embodiments, the cellis a rodent cell. In some embodiments, the cell is a human cell. In someembodiments, the cell is a liver cell. In certain embodiments, the cellis a human liver cell. In some embodiments, the liver cell is ahepatocyte. In some embodiments, the hepatocyte is a human hepatocyte.In some embodiments, the liver cell is a stem cell. In some embodiments,the human liver cell is a liver sinusoidal endothelial cell (LSEC). Insome embodiments, the human liver cell is a Kupffer cell. In someembodiments, the human liver cell is a hepatic stellate cell. In someembodiments, the human liver cell is a tumor cell. In some embodiments,the human liver cell is a liver stem cell. In some embodiments, the cellcomprises ApoE-binding receptors. In some embodiments, engineered cellsare provided; for example an engineered cell can be derived from any oneof the cell types as described herein. Such engineered cells can beproduced according to the methods described herein. In some embodiments,the engineered cell resides within a tissue or organ, e.g., a liverwithin a subject.

Target Sequences

The present disclosure provides active agents or therapeutic agents,such as genome editing compositions, and methods and compositions fortargeted delivery thereof. The therapeutic agents described herein maycomprise genome editing composition directed to and modify, alter, orcleave a target sequence on a target nucleic acid molecule. For example,the active agent may comprise a nucleic acid or a nucleic acid-proteincomplex capable of effecting a modification to a target sequence.

The target sequence may be a DNA sequence or a RNA sequence. In someembodiments, the active agent or therapeutic agent may comprise a RNAinterference factor. In some embodiments, the active agent may comprisea siRNA, shRNA, antisense oligonucleotide, microRNA, anti-microRNA orantimir, supermir, antagomir, ribozyme, triplex-forming oligonucleotide,decoy oligonucleotide, splice-switching oligonucleotide,immunostimulatory oligonucleotide, RNA activator, or a Ul adaptor. Theactive agent may recognize the target sequence and mediate cleavageand/or degradation of the target sequence. In some embodiments, theactive or therapeutic agent may comprise a guide RNA. The guide RNA maybe complexed with a nucleic acid guided programmable nuclease, such as aCRISPR enzyme, such as a Cas9, or a fusion protein thereof furthercomprising a functional domain. The target sequence may be recognized bythe nucleic acid guided programmable nuclease domain. The targetsequence may be cleaved by the nucleic acid guided programmable nucleasedomain and/or modified by the functional domain, such as a deaminasedomain, a methylase domain, a methyltransferase domain, an activationdomain, a repressor domain, a nuclease domain, a transposase domain, ora recombinase domain. In some embodiments, a Cas9 protein may bedirected by a guide RNA to a target sequence of a target nucleic acidmolecule, where the guide RNA hybridizes with and the Cas proteincleaves the target sequence. In some embodiments, the target sequencemay be complementary to the targeting sequence of the guide RNA. In someembodiments, the degree of complementarity between a targeting sequenceof a guide RNA and its corresponding target sequence may be about 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. Insome embodiments, the target sequence and the targeting sequence of theguide RNA may be 100% complementary. In other embodiments, the targetsequence and the targeting sequence of the guide RNA may contain atleast one mismatch. For example, the target sequence and the targetingsequence of the guide RNA may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10mismatches. In some embodiments, the target sequence and the targetingsequence of the guide RNA may contain 1-6 mismatches. In someembodiments, the target sequence and the targeting sequence of the guideRNA may contain 5 or 6 mismatches.

The length of the target sequence may depend on the nuclease systemused. For example, the target sequence for a CRISPR/Cas system maycomprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more than 50nucleotides in length. In some embodiments, the target sequence maycomprise 18-24 nucleotides in length. In some embodiments, the targetsequence may comprise 19-21 nucleotides in length. In some embodiments,the target sequence may comprise 20 nucleotides in length. When nickasesare used, the target sequence may comprise a pair of target sequencesrecognized by a pair of nickases on opposite strands of the DNAmolecule.

In some embodiments, the active or therapeutic agent may comprise ameganuclease system. the target sequence for a meganuclease may comprise12-40 or more nucleotides in length. When ZFNs are used, the targetsequence may comprise two half target sequences recognized by a pair ofZFNs on opposite strands of the DNA molecule, with an interconnectingsequence in between. In some embodiments, each half target sequence forZFNs may independently comprise 9, 12, 15, 18, or more nucleotides inlength. In some embodiments, the interconnecting sequence for ZFNs maycomprise 4-20 nucleotides in length. In some embodiments, theinterconnecting sequence for ZFNs may comprise 5-7 nucleotides inlength.

When TALENs are used, the target sequence may similarly comprise twohalf target sequences recognized by a pair of TALENs on opposite strandsof the DNA molecule, with an interconnecting sequence in between. Insome embodiments, each half target sequence for TALENs may independentlycomprise 10-20 or more nucleotides in length. In some embodiments, theinterconnecting sequence for TALENs may comprise 4-20 nucleotides inlength. In some embodiments, the interconnecting sequence for TALENs maycomprise 12-19 nucleotides in length.

In some embodiments, the target sequence may be adjacent to aprotospacer adjacent motif (PAM), a short sequence recognized by aCRISPR/Cas complex. The protospacer adjacent motif, or PAM, is essentialfor target binding for CRISPR/Cas complexes. Typically, a PAM is a 2-6base pair DNA sequence immediately following the DNA target sequence ofthe Cas nuclease. The PAM may be a 5′ PAM or a 3′ PAM. The exactsequence of PAM depends on the type of Cas protein. For example, atypical SpCas9 binding requires a 3′-NGG-S′ PAM, also known as acanonical PAM, where the N is any one of A, G, C, or T. A SpCas9 withcertain amino acid substitutions, e.g. D1135E, R1335Q, G1218R, and/orT1337R can recognize a NGA PAM or a NGCG PAM. A SaCas9 binding requiresa 3′-NNGRRT-5′ PAM. A SaCas9 with certain amino substitutions, e.g.,K781E, K697N, H1014R, can recognize a NNNRRT PAM.

In some embodiments, the PAM may be adjacent to or within 1, 2, 3, or 4,nucleotides of the 3′ end of the target sequence. The length and thesequence of the PAM may depend on the Cas9 protein used. For example,the PAM may be selected from a consensus or a particular PAM sequencefor a specific Cas9 protein or Cas9 ortholog, including those disclosedin FIG. 1 of Ran et al., Nature, 520: 186-191 (2015), which isincorporated herein by reference. In some embodiments, the PAM maycomprise 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length.Non-limiting exemplary PAM sequences include NGG, NGGNG, NG, NAAAAN,NNAAAAW, NNNNACA, GNNNCNNA, and NNNNGATT (wherein N is defined as anynucleotide, and W is defined as either A or T). In some embodiments, thePAM sequence may be NGG. In some embodiments, the PAM sequence may beNGGNG. In some embodiments, the PAM sequence may be NNAAAAW. Additionalevolved Cas variants and PAM sequences as described in Hu et al.,Evolved Cas9 variants with broad PAM compatibility and high DNAspecificity, Nature 2018 556(7699): 57-63 is incorporated herein in itsentirety.

The target nucleic acid molecule may be any DNA or RNA molecule that isendogenous or exogenous to a cell. As used herein, the term “endogenoussequence” refers to a sequence that is native to the cell. The term“exogenous sequence” refers to a sequence that is not native to a cell,or a sequence whose native location in the genome of the cell is in adifferent location. In some embodiments, the target nucleic acidmolecule may be a plasmid, a genomic DNA, or a chromosome from a cell orin the cell. In some embodiments, the target sequence of the targetnucleic acid molecule may be a genomic sequence from a cell or in thecell. In some embodiments, the cell may be a prokaryotic cell. In otherembodiments, the cell may be a eukaryotic cell. In some embodiments, theeukaryotic cell may be a mammalian cell. In some embodiments, theeukaryotic cell may be a rodent cell. In some embodiments, theeukaryotic cell may be a human cell. In some embodiments, the eukaryoticcell may be a liver cell. In some embodiments, the eukaryotic cell maybe a hepatocyte. In some embodiments, the eukaryotic cell may be aparenchymal cell, a sinusoidal endothelial cell, a phagocytic Kupffercell, or a stellate cell. In further embodiments, the target sequencemay be a viral sequence. In yet other embodiments, the target sequencemay be a synthesized sequence. In some embodiments, the target sequencemay be on a eukaryotic chromosome, such as a human chromosome.

In some embodiments, the target sequence may be located in a codingsequence of a gene, an intron sequence of a gene, a transcriptionalcontrol sequence of a gene, a translational control sequence of a gene,or a non-coding sequence between genes. In some embodiments, the genemay be a protein coding gene. In other embodiments, the gene may be anon-coding RNA gene. In some embodiments, the target sequence maycomprise all or a portion of a disease-associated gene. In someembodiments, the target sequence may comprise all or a portion of a geneassociated with a coronary disease. In some embodiments, the targetsequence may comprise at least a portion of a gene encoding anapolipoprotein. In some embodiments, the target sequence may comprise atleast a portion of a gene selected from PCSK9, ANGPTL3, APOC3, LPA,APOB, MTP, ANGPTL4, ANGPTL8, APOA5, APOE, LDLR, IDOL, NPC1L1, ASGR1,TM6SF2, GALNT2, GCKR, LPL, MLXIPL, SORT1, TRIB1, MARC1, ABCG5, andABCG8.

In some embodiments, contacting a target sequences with the genomeediting composition described herein leads to a base editing eventwithin or adjacent to the target sequence. For example, a target base(e.g. a C base) within or adjacent to a target sequence may be convertedto a T base as the result of contact with the genome editing compositionas disclosed in the present disclosure comprising a fusion proteincomprising a nucleic acid guided nuclease domain and a deaminase domain.In some embodiments, the target base is located upstream (5′ end of) ofthe PAM. In some embodiments, the target base is located at a position1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 base pairs upstream (5′end of) the PAM. In some embodiments, the target base is located at aposition within 13 to 17 base pairs upstream (5′ end) of the PAM. Insome embodiments, the target base is located at a position outside of 13to 17 base pairs upstream (5′ end) of the PAM. In some embodiments, thetarget base pair is located at a position 10-15 base pairs upstream (5′end) of the PAM. In some embodiments, the target base is located at aposition 11-12 base pairs upstream of the PAM. In some embodiments, thetarget base is 11 base pairs upstream (5′ end) of the PAM. In someembodiments, the target base is located in the coding region (e.g., anexon) of the target sequence, e.g. the ANGPTL3 encoding polynucleotide(e.g., the ANGPTL3 gene locus). For example, conversion of a base in thecoding region of the ANGPTL3 gene locus may result in an amino acidchange in the ANGPTL3 protein sequence, i.e., a mutation. In someembodiments, the mutation is a loss of function mutation. In someembodiments, a mutation may introduce a pre-mature stop codon into thecoding region the target sequence, e.g. coding region of the ANGPTL3gene. In some embodiments, a loss-of-function mutation is a naturallyoccurring loss-of-function mutation. In some embodiments, the mutationis located in the coding region of the PCSK9 gene, e.g. a G106R, L253F,A443T, R93C, G24D, S47F, R46H, S 153N, or H193Y mutation. In someembodiments, the loss-of-function mutation introduces a pre-mature stopcodon into the coding region of the ANGPTL3 gene. In some embodiments, aloss of function mutation may be introduced into the coding region of aAPOC3 gene, e.g. a R19X mutation. In some embodiments, a loss offunction mutation may be introduced into a Low-Density LipoproteinReceptor (LDL-R) protein. In some embodiments, a loss of functionmutation may be introduced into a Inducible Degrader of the LDL Receptor(IDOL) protein.

In some embodiments, a target sequence is located in a non-coding regionof the target sequence, e.g., in an intron or a splicing site of atarget gene. In some embodiments, a target sequence is located in asplicing site and the editing of such target base causes alternativesplicing of the target gene mRNA. In some embodiments, the alternativesplicing leads to leading to loss-of-function mutants. In someembodiments, the alternative splicing leads to introduction of apremature stop codon or a frameshift in the target mRNA, resulting intruncated, unstable, or folding-defective polypeptides. In someembodiments, stop codons may be introduced into the coding sequence of aapolipoprotein encoding gene upstream of the normal stop codon (referredto as a “premature stop codon”). In some embodiments, stop codons may beintroduced into the coding region of the target gene. Premature stopcodons cause premature translation termination, in turn resulting intruncated and nonfunctional proteins and induces rapid degradation ofthe mRNA via the non-sense mediated mRNA decay pathway. See, e.g., Bakeret al., Current Opinion in Cell Biology 16 (3): 293-299, 2004; Chang etal, Annual Review of Biochemistry 76: 51-74, 2007; and Behm-Ansmant etah, Genes & Development 20 (4): 391-398, 2006, each of which isincorporated herein by reference. The genome editing compositiondescribed herein may be used to introduce multiple editing events to thetarget sequence. For example, the genome editing composition maycomprise a nucleic acid guide programmable nuclease that induces doublestrand breaks, deletions, insertions, frameshift, reversions, or otheralterations in the target gene. For example, the genome editingcomposition may comprise a nucleic acid guided programmablenuclease-deaminase fusion protein that can convert several amino acidsto create a stop codon (e.g., TAA, TAG, or TGA).

In some embodiments, simultaneous introduction of mutations into morethan one protein factors in the LDL-mediated cholesterol clearancepathway are provided. For example, in some embodiments, a mutation maybe simultaneously introduced into one or more, preferably at least two,of ANGPTL3, PCSK9, LDLR, APOB, APOE, IDOL, and other LDL-mediatedpathway involved genes. In some embodiments, a loss-of-function mutationmay be simultaneously introduced into one or more, preferably at leasttwo, of ANGPTL3, PCSK9, APOB, and another LDL-mediated pathway involvedgene. In some embodiments, mutations may be simultaneously introducedinto ANGPTL3, PCSK9, LDLR, and IDOL. To simultaneously introduce ofloss-of-function mutations into more than one protein, multiple guidenucleotide sequences are used.

In some embodiments, the target sequence may be located in a non-genicfunctional site in the genome that controls aspects of chromatinorganization, such as a scaffold site or locus control region. In someembodiments, the target sequence may be a genetic safe harbor site,i.e., a locus that facilitates safe genetic modification.

Templates

In some embodiments, at least one template may be provided as asubstrate during the repair of the cleaved target nucleic acid molecule.In some embodiments, the template may be used in homologousrecombination, such as, e.g., high-fidelity homologous recombination. Insome embodiments, the homologous recombination may result in theintegration of the template sequence into the target nucleic acidmolecule. In some embodiments, a single template or multiple copies ofthe same template may be provided. In other embodiments, two or moretemplates may be provided such that homologous recombination may occurat two or more target sites. For example, different templates may beprovided to repair a single gene in a cell, or two different genes in acell. In some embodiments, the different templates may be provided inindependent copy numbers.

In some embodiments, the template may be used in homology-directedrepair, requiring DNA strand invasion at the site of the cleavage in thenucleic acid. In some embodiments, the homology-directed repair mayresult in the copying of the template sequence into the target nucleicacid molecule. In some embodiments, a single template or multiple copiesof the same template may be provided. In other embodiments, two or moretemplates having different sequences may be inserted at two or moresites by homology-directed repair. For example, different templates maybe provided to repair a single gene in a cell, or two different genes ina cell. In some embodiments, the different templates may be provided inindependent copy numbers.

In some embodiments, the template may be incorporated into the cleavednucleic acid as an insertion mediated by non-homologous end joining. Insome embodiments, the template sequence has no similarity to the nucleicacid sequence near the cleavage site. In some embodiments, the templatesequence (e.g., the coding sequence in the template) has no similarityto the nucleic acid sequence near the cleavage site. The templatesequence may be flanked by target sequences that may have similar oridentical sequence(s) to a target sequence near the cleavage site. Insome embodiments, a single template or multiple copies of the sametemplate may be provided. In other embodiments, two or more templateshaving different sequences may be inserted at two or more sites bynon-homologous end joining. For example, different templates may beprovided to insert a single template in a cell, or two differenttemplates in a cell. In some embodiments, the different templates may beprovided in independent copy numbers.

In some embodiments, the template sequence may correspond to anendogenous sequence of a target cell. In some embodiments, theendogenous sequence may be a genomic sequence of the cell. In someembodiments, the endogenous sequence may be a chromosomal orextrachromosomal sequence. In some embodiments, the endogenous sequencemay be a plasmid sequence of the cell. In some embodiments, the templatesequence may be substantially identical to a portion of the endogenoussequence in a cell at or near the cleavage site, but comprise at leastone nucleotide change. In some embodiments, the repair of the cleavedtarget nucleic acid molecule with the template may result in a mutationcomprising an insertion, deletion, or substitution of one or morenucleotides of the target nucleic acid molecule. In some embodiments,the mutation may result in one or more amino acid changes in a proteinexpressed from a gene comprising the target sequence. In someembodiments, the mutation may result in one or more nucleotide changesin an RNA expressed from the target gene. In some embodiments, themutation may alter the expression level of the target gene. In someembodiments, the mutation may result in increased or decreasedexpression of the target gene. In some embodiments, the mutation mayresult in gene knockdown. In some embodiments, the mutation may resultin gene knockout. In some embodiments, the repair of the cleaved targetnucleic acid molecule with the template may result in replacement of anexon sequence, an intron sequence, a transcriptional control sequence, atranslational control sequence, or a non-coding sequence of the targetgene.

In other embodiments, the template sequence may comprise an exogenoussequence. In some embodiments, the exogenous sequence may comprise aprotein or RNA coding sequence operably linked to an exogenous promotersequence such that, upon integration of the exogenous sequence into thetarget nucleic acid molecule, the cell is capable of expressing theprotein or RNA encoded by the integrated sequence. In other embodiments,upon integration of the exogenous sequence into the target nucleic acidmolecule, the expression of the integrated sequence may be regulated byan endogenous promoter sequence. In some embodiments, the exogenoussequence may be a chromosomal or extrachromosomal sequence. In someembodiments, the exogenous sequence may provide a cDNA sequence encodinga protein or a portion of the protein. In yet other embodiments, theexogenous sequence may comprise an exon sequence, an intron sequence, atranscriptional control sequence, a translational control sequence, or anon-coding sequence. In some embodiments, the integration of theexogenous sequence may result in gene knock-in.

The template may be of any suitable length. In some embodiments, thetemplate may comprise 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000,1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, or morenucleotides in length. In some embodiments, the template may comprise anucleotide sequence that is complementary to a portion of the targetnucleic acid molecule comprising the target sequence (i.e., a “homologyarm”). In some embodiments, a homology arm may comprise 10, 15, 20, 25,50, 75, 100, 150, 200, 500, 1000, 1500, 2000, 2500, 3000 or morenucleotides in length. In some embodiments, the template may comprise ahomology arm that is complementary to the sequence located upstream ordownstream of the cleavage site on the target nucleic acid molecule. Insome embodiments, the template may comprise a first nucleotide sequenceand a second homology arm that are complementary to the sequenceslocated upstream and downstream of the cleavage site, respectively.Where a template contains two homology arms, each arm can be the samelength or different lengths, and the sequence between the homology armscan be substantially similar or identical to the target sequence betweenthe homology arms, or be entirely unrelated. In some embodiments, thedegree of complementarity between the first nucleotide sequence on thetemplate and the sequence upstream of the cleavage site, and between thesecond nucleotide sequence on the template and the sequence downstreamof the cleavage site, may permit homologous recombination, such as,e.g., high-fidelity homologous recombination, between the template andthe target nucleic acid molecule. In some embodiments, the degree ofcomplementarity may be about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the degree ofcomplementarity may be about 95%, 97%, 98%, 99%, or 100%. In someembodiments, the degree of complementarity may be about 98%, 99%, or100%. In some embodiments, the degree of complementarity may be 100%. Insome embodiments, for example those described herein where a template isincorporated into the cleaved nucleic acid as an insertion mediated bynon-homologous end joining, the template has no homology arms. In someembodiments, a template having no homology arms comprises targetsequences flanking one or both ends of the template sequence, e.g., asdescribed herein. In some embodiments, a template having no homologyarms comprises target sequences flanking both ends of the templatesequence. In some embodiments, a target sequence flanking the end of thetemplate sequence is about 10-50 nucleotides. In some embodiments, atarget sequence flanking the end of the template sequence is about 10-20nucleotides, about 15-20 nucleotides, about 20-25 nucleotides, or about20-30 nucleotides. In some embodiments, a target sequence flanking theend of the template sequence is about 17-23 nucleotides. In someembodiments, a target sequence flanking the end of the template sequenceis about 20 nucleotides.

In some embodiments, a nucleic acid molecule is expressed from thetemplate if homologous recombination occurs between the template and thegenomic sequence. In some embodiments, for example, the template doesnot have a promoter for expressing the nucleic acid molecule and/or theATG transcriptional start site is removed from the coding sequence.

Delivery

Provided herein are methods and compositions for editing a nucleic acidmolecule in a cell with a nuclease system and targeted delivery thereof.In some embodiments, the nucleic acid comprises a nucleic acid sequenceencoding a gene. In some embodiments, the nucleic acid comprises anucleic acid sequence encoding a gene associated with a disease ordisorder.

The active agents comprising nucleic acids described herein, e.g.modified guide RNAs, may be conjugated with one or more targetingmoieties for targeted delivery to desired in vivo locations. The guideRNA conjugates or guide RNA-protein complex conjugates may be introducedinto the cell via any methods known in the art, such as, e.g., viral orbacteriophage infection, transfection, conjugation, protoplast fusion,lipofection, lipid particle or vesicle transduction, electroporation,calcium phosphate precipitation, polyethyleneimine (PEI)-mediatedtransfection, DEAE-dextran-mediated transfection, liposome-mediatedtransfection, e.g. transfection mediated by cationic liposomes, particlegun technology, calcium phosphate precipitation, shear-driven cellpermeation, fusion to a cell-penetrating peptide followed by cellcontact, microinjection, and nanoparticle-mediated delivery. In someembodiments, the nuclease system may be introduced into the cell viaviral infection. In some embodiments, the nuclease system may beintroduced into the cell via bacteriophage infection. Liposomes mayinclude those formed from 1,2-dioleyloxy-N,N-dimethylaminopropane(DODMA) liposomes, DiLa2 liposomes from Marina Biotech (Bothell, Wash.),1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA),2,2-dilinoleyl-4-(2-dimethylaminoethyl)[1,3]-dioxolane (DLin-KC2-DMA),and liposomes which may deliver small molecule drugs such as, but notlimited to, DOXIL® (from Janssen Biotech, Inc. (Horsham, Pa.)).

In some embodiments, the methods and compositions provided herein maycomprise introducing a vector system described herein into a cell. Insome embodiments, the vector system encodes the nuclease system in wholeor in part. In some embodiments, the vector system comprises one, two,three, or more vectors. In some embodiments, the introduction of thevector system into the cell may result in a stable cell line having theedited nucleic acid molecule while the vectors are lost, e.g., targetedfor self-destruction. In some embodiments, the cell is a eukaryoticcell. Non-limiting examples of eukaryotic cells include yeast cells,plant cells, insect cells, cells from an invertebrate animal, cells froma vertebrate animal, mammalian cells, rodent cells, mouse cells, ratcells, and human cells. In some embodiments, the eukaryotic cell may bea mammalian cell. In some embodiments, the eukaryotic cell may be arodent cell. In some embodiments, the eukaryotic cell may be a humancell. Similarly, the target sequence may be from any such cells or inany such cells.

In some embodiments, the polynucleotides or oligonucleotides providedherein, for example guide RNAs or mRNAs, may be formulated in a lipidvesicle which may have crosslinks between functionalized lipid bilayers,or lipid-polycation complex. The liposome formulation may be influencedby, but not limited to, the selection of the cationic lipid component,the degree of cationic lipid saturation, the nature of the PEGylation,ratio of all components and biophysical parameters such as size, or polycationic composition. In one embodiment, pharmaceutical compositionsdescribed herein may include, without limitation, liposomes such asthose formed from the synthesis of stabilized plasmid-lipid particles(SPLP) or stabilized nucleic acid lipid particle (SNALP) that have beenpreviously described and shown to be suitable for oligonucleotidedelivery in vitro and in vivo. The lipid nanoparticles may be engineeredto alter the surface properties of particles so the lipid nanoparticlesmay penetrate the mucosal barrier. Mucus is located on mucosal tissuesuch as, but not limited to, oral (e.g., the buccal and esophagealmembranes and tonsil tissue), ophthalmic, gastrointestinal (e.g.,stomach, small intestine, large intestine, colon, rectum), respiratory(e.g., nasal, pharyngeal, tracheal and bronchial membranes), genital(e.g., vaginal, cervical and urethral membranes). The formulations mayuse nanoparticles larger than 10-200 nm which are preferred for higherdrug encapsulation efficiency and the ability to provide the sustaineddelivery of a wide array of drugs which had been thought to be too largeto rapidly diffuse through mucosal barriers. The dynamic transport ofnanoparticles may be measured using fluorescence recovery afterphotobleaching (FRAP) and high resolution multiple particle tracking(MPT). The formulations can be made for controlled release and/ortargeted delivery. The lipid nanoparticle engineered to penetrate mucusmay include surface altering agents such as, but not limited to, mRNA,anionic protein (e.g., bovine serum albumin), surfactants (e.g.,cationic surfactants such as for example dimethyldioctadecylammoniumbromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleicacids, polymers (e.g., heparin, polyethylene glycol and poloxamer),mucolytic agents (e.g., N-acetylcysteine, mugwort, bromelain, papain,clerodendrum, acetylcysteine, bromhexine, carbocisteine, eprazinone,mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin,gelsolin, thymosin (34, dornase alfa, neltenexine, erdosteine) andvarious DNases including rhDNase. The surface altering agent may beembedded or enmeshed in the particle's surface or disposed or dispersed(e.g., by coating, adsorption, covalent linkage, or other process) onthe surface of the lipid nanoparticle.

In a further embodiment, guide RNA of the present disclosure and theCRISPR system may be formulated as a lipoplex, such as, withoutlimitation, the ATUPLEX™ system, the DACC system, the DBTC system andother siRNA-lipoplex technology. The liposomes, lipoplexes, or lipidnanoparticles may be used to improve the efficacy of the modified guideRNAs for example by increasing cell transfection, increasing thetranslation of encoded protein or increasing the stability. A cellpenetrating peptide may be used with the pharmaceutical formulations ofthe present disclosure such as a cell-penetrating peptide sequenceattached to polycations that facilitates delivery to the intracellularspace, e.g., HIV-derived TAT peptide, penetratins, transportans, or hCTderived cell-penetrating peptides. In another embodiment, lipidnanoparticles which target specific cell types may be used.Alternatively, the lipid nanoparticle may be encapsulated into anypolymer or hydrogel known in the art which may form a gel when injectedinto a subject. As another non-limiting example, the lipid nanoparticlemay be encapsulated into a polymer matrix which may be biodegradable. Inyet another embodiment, the pharmaceutical compositions may be sustainedrelease formulations. In a further embodiment, the sustained releaseformulations may be for subcutaneous delivery. Sustained releaseformulations may include, but are not limited to, PLGA microspheres,ethylene vinyl acetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics,Inc. Alachua, Fla.), HYLENEX® (Halozyme Therapeutics, San Diego,Calif.), surgical sealants such as fibrinogen polymers (Ethicon Inc.Cornelia, Ga.), TISSELL® (Baxter International, Inc Deerfield, Ill.),PEG-based sealants, and COSEAL® (Baxter International, Inc Deerfield,Ill.).

In some embodiments, the nucleic acids as described herein, such as aguide RNA, may be complexed with a CRISPR enzyme. In some embodiments, apart or all of the complex may be delivered via a vector systemcomprising one or more vectors. In some embodiments, the vector may be aDNA vector. In other embodiments, the vector may be an RNA vector. Insome embodiments, the RNA vector may be an mRNA, e.g. an mRNA thatencodes a nuclease such as Cas9. In some embodiments, the vector may becircular. In other embodiments, the vector may be linear. Non-limitingexemplary vectors include plasmids, phagemids, cosmids, artificialchromosomes, minichromosomes, transposons, viral vectors, and expressionvectors. In some embodiments, the nuclease is provided by an RNA vector,e.g., as mRNA, and the template is provided by a viral vector. In someembodiments, the vector may be a viral vector. In some embodiments, theviral vector may be genetically modified from its wild-type counterpart.For example, the viral vector may comprise an insertion, deletion, orsubstitution of one or more nucleotides to facilitate cloning or suchthat one or more properties of the vector is changed. Such propertiesmay include packaging capacity, transduction efficiency, immunogenicity,genome integration, replication, transcription, and translation. In someembodiments, a portion of the viral genome may be deleted such that thevirus is capable of packaging exogenous sequences having a larger size.In some embodiments, the viral vector may have an enhanced transductionefficiency. In some embodiments, the immune response induced by thevirus in a host may be reduced. In some embodiments, viral genes (suchas, e.g., integrase) that promote integration of the viral sequence intoa host genome may be mutated such that the virus becomesnon-integrating. In some embodiments, the viral vector may bereplication defective. In some embodiments, the viral vector maycomprise exogenous transcriptional or translational control sequences todrive expression of coding sequences on the vector. In some embodiments,the virus may be helper-dependent. For example, the virus may need oneor more helper virus to supply viral components (such as, e.g., viralproteins) required to amplify and package the vectors into viralparticles. In such a case, one or more helper components, including oneor more vectors encoding the viral components, may be introduced into ahost cell along with the vector system described herein. In otherembodiments, the virus may be helper-free. For example, the virus may becapable of amplifying and packaging the vectors without any helpervirus. In some embodiments, the vector system described herein may alsoencode the viral components required for virus amplification andpackaging.

Non-limiting exemplary viral vectors include adeno-associated virus(AAV) vector, lentivirus vectors, adenovirus vectors, herpes simplexvirus (HSV-1) vectors, bacteriophage T4, baculovirus vectors, andretrovirus vectors. In some embodiments, the viral vector may be an AAVvector. In other embodiments, the viral vector may a lentivirus vector.In some embodiments, the lentivirus may be non-integrating. In someembodiments, the viral vector may be an adenovirus vector. In someembodiments, the adenovirus may be a high-cloning capacity or “gutless”adenovirus, where all coding viral regions apart from the 5′ and 3′inverted terminal repeats (ITRs) and the packaging signal (Ψ) aredeleted from the virus to increase its packaging capacity. In yet otherembodiments, the viral vector may be an HSV-1 vector. In someembodiments, the HSV-1-based vector is helper dependent, and in otherembodiments it is helper independent. For example, an amplicon vectorthat retains only the packaging sequence requires a helper virus withstructural components for packaging, while a 30 kb-deleted HSV-1 vectorthat removes non-essential viral functions does not require helpervirus. In additional embodiments, the viral vector may be bacteriophageT4. In some embodiments, the bacteriophage T4 may be able to package anylinear or circular DNA or RNA molecules when the head of the virus isemptied. In further embodiments, the viral vector may be a baculovirusvector. In yet further embodiments, the viral vector may be a retrovirusvector. In embodiments using AAV or lentiviral vectors, which havesmaller cloning capacity, it may be necessary to use more than onevector to deliver all the components of a vector system as disclosedherein. For example, one AAV vector may contain sequences encoding aCas9 protein, while a second AAV vector may contain one or more guidesequences and one or more copies of template.

In certain embodiments, a viral vector may be modified to target aparticular tissue or cell type. For example, viral surface proteins maybe altered to decrease or eliminate viral protein binding to its naturalcell surface receptor(s). In some embodiments, the vector may bemodified for liver specific delivery. The surface proteins may also beengineered to interact with a receptor specific to a desired cell type.Viral vectors may have altered host tropism, including limited orredirected tropism. In some embodiments, the viral vector may beengineered to express or display a first binding moiety. The firstbinding moiety may be fused to a viral surface protein or glycoprotein,conjugated to a virus, chemically crosslinked to a virion, bound to avirus envelope, or joined to a viral vector by any other suitablemethod. The first binding moiety is capable of binding to a secondbinding moiety, which may be used to direct the virus to a desired celltype. In some embodiments, the first binding moiety is avidin,streptavidin, neutravidin, captavidin, or another biotin-binding moiety,and the second binding moiety is biotin or an analog thereof. Abiotinylated targeting agent may then be bound to the avidin on theviral vector and used to direct the virus to a desired cell type. Forexample, a T4 vector may be engineered to display a biotin-bindingmoiety on one or more of its surface proteins. The cell-specificity ofsuch a T4 vector may then be altered by binding a biotinylated antibodyor ligand directed to a cell of choice. In alternate embodiments, thefirst and second binding moieties are hapten and an anti-hapten bindingprotein; digoxigenin and an anti-digoxigenin binding protein;fluorescein and an anti-fluorescein binding protein; or any othersuitable first and second binding moieties that are binding partners.

In some embodiments, the vector may be capable of driving expression ofone or more coding sequences in a cell. In some embodiments, the cellmay be a prokaryotic cell, such as, e.g., a bacterial cell. In someembodiments, the cell may be a eukaryotic cell, such as, e.g., a yeast,plant, insect, or mammalian cell. In some embodiments, the eukaryoticcell may be a mammalian cell. In some embodiments, the eukaryotic cellmay be a rodent cell. In some embodiments, the eukaryotic cell may be ahuman cell. Suitable promoters to drive expression in different types ofcells are known in the art. In some embodiments, the promoter may bewild-type. In other embodiments, the promoter may be modified for moreefficient or efficacious expression. In yet other embodiments, thepromoter may be truncated yet retain its function. For example, thepromoter may have a normal size or a reduced size that is suitable forproper packaging of the vector into a virus.

In some embodiments, the vector may comprise a nucleotide sequenceencoding the nuclease described herein. In some embodiments, the vectorsystem may comprise one copy of the nucleotide sequence encoding thenuclease. In other embodiments, the vector system may comprise more thanone copy of the nucleotide sequence encoding the nuclease. In someembodiments, the nucleotide sequence encoding the nuclease may beoperably linked to at least one transcriptional or translational controlsequence. In some embodiments, the nucleotide sequence encoding thenuclease may be operably linked to at least one promoter. In someembodiments, the nucleotide sequence encoding the nuclease may beoperably linked to at least one transcriptional or translational controlsequence.

In some embodiments, the promoter may be constitutive, inducible, ortissue-specific. In some embodiments, the promoter may be a constitutivepromoter. Non-limiting exemplary constitutive promoters includecytomegalovirus immediate early promoter (CMV), simian virus (SV40)promoter, adenovirus major late (MLP) promoter, Rous sarcoma virus (RSV)promoter, mouse mammary tumor virus (MMTV) promoter, phosphoglyceratekinase (PGK) promoter, elongation factor-alpha (EF1α) promoter,ubiquitin promoters, actin promoters, tubulin promoters, immunoglobulinpromoters, a functional fragment thereof, or a combination of any of theforegoing. In some embodiments, the promoter may be a CMV promoter. Insome embodiments, the promoter may be a truncated CMV promoter. In otherembodiments, the promoter may be an EF1α promoter. In some embodiments,the promoter may be an inducible promoter. Non-limiting exemplaryinducible promoters include those inducible by heat shock, light,chemicals, peptides, metals, steroids, antibiotics, or alcohol. In someembodiments, the inducible promoter may be one that has a low basal(non-induced) expression level, such as, e.g., the Tet-On® promoter(Clontech). In some embodiments, the promoter may be a tissue-specificpromoter. In some embodiments, the tissue-specific promoter isexclusively or predominantly expressed in liver tissue. Non-limitingexemplary tissue-specific promoters include B29 promoter, CD14 promoter,CD43 promoter, CD45 promoter, CD68 promoter, desmin promoter, elastase-1promoter, endoglin promoter, fibronectin promoter, Flt-1 promoter, GFAPpromoter, GPIIb promoter, ICAM-2 promoter, INF-β promoter, Mb promoter,Nphsl promoter, OG-2 promoter, SP-B promoter, SYN1 promoter, and WASPpromoter.

In some embodiments, the vector may encode a Cas protein or a portion ofa Cas protein, such as a Cas9 protein or Cpf1 protein. The vector systemmay further comprise a vector comprising a nucleotide sequence encodingthe guide RNA described herein. In some embodiments, the vector systemmay comprise one copy of the guide RNA. In other embodiments, the vectorsystem may comprise more than one copy of the guide RNA. In embodimentswith more than one guide RNA, the guide RNAs may be non-identical suchthat they target different target sequences, or have other differentproperties, such as activity or stability within the Cas9 RNP complex.In some embodiments, the nucleotide sequence encoding the guide RNA maybe operably linked to at least one transcriptional or translationalcontrol sequence. In some embodiments, the nucleotide sequence encodingthe guide RNA may be operably linked to at least one promoter. In someembodiments, the promoter may be recognized by RNA polymerase III (PolIII). Non-limiting examples of Pol III promoters include U6, H1 and tRNApromoters. In some embodiments, the nucleotide sequence encoding theguide RNA may be operably linked to a mouse or human U6 promoter. Inother embodiments, the nucleotide sequence encoding the guide RNA may beoperably linked to a mouse or human H1 promoter. In some embodiments,the nucleotide sequence encoding the guide RNA may be operably linked toa mouse or human tRNA promoter. In embodiments with more than one guideRNA, the promoters used to drive expression may be the same ordifferent. In some embodiments, the nucleotide encoding the crRNA of theguide RNA and the nucleotide encoding the tracr RNA of the guide RNA maybe provided on the same vector. In some embodiments, the nucleotideencoding the crRNA and the nucleotide encoding the tracr RNA may bedriven by the same promoter. In some embodiments, the crRNA and tracrRNA may be transcribed into a single transcript. For example, the crRNAand tracr RNA may be processed from the single transcript to form adouble-molecule guide RNA. Alternatively, the crRNA and tracr RNA may betranscribed into a single-molecule guide RNA. In other embodiments, thecrRNA and the tracr RNA may be driven by their corresponding promoterson the same vector. In yet other embodiments, the crRNA and the tracrRNA may be encoded by different vectors.

In some embodiments, the vector system may further comprise a vectorcomprising the template described herein. In some embodiments, thevector system may comprise one copy of the template. In otherembodiments, the vector system may comprise more than one copy of thetemplate. In some embodiments, the vector system may comprise 2, 3, 4,5, 6, 7, 8, 9, 10, or more copies of the template. In some embodiments,the vector system may comprise 4, 5, 6, 7, 8, or more copies of thetemplate. In some embodiments, the vector system may comprise 5, 6, 7,or more copies of the template. In some embodiments, the vector systemmay comprise 6 copies of the template. The multiple copies of thetemplate may be located on the same or different vectors. The multiplecopies of the template may also be adjacent to one another, or separatedby other nucleotide sequences or vector elements. In other embodiments,two or more templates may be provided such that homologous recombinationmay occur at two or more target sites. For example, different templatesmay be provided to repair a single gene in a cell, or two differentgenes in a cell. In some embodiments, the different templates may beprovided in independent copy numbers.

A vector system may comprise 1-3 vectors. In some embodiments, thevector system may comprise one single vector. In other embodiments, thevector system may comprise two vectors. In additional embodiments, thevector system may comprise three vectors.

In some embodiments, the nucleotide sequence encoding the nuclease andthe template may be located on the same or separate vectors. In someembodiments, the nucleotide sequence encoding the nuclease and thetemplate may be located on the same vector. In some embodiments, thenucleotide sequence encoding the nuclease and the template may belocated on separate vectors. The sequences may be oriented in the sameor different directions and in any order on the vector.

In some embodiments, the nucleotide sequence encoding a Cas9 protein anda template may be located on the same or separate vectors. In someembodiments, all of the sequences may be located on the same vector. Insome embodiments, two or more sequences may be located on the samevector. The sequences may be oriented in the same or differentdirections and in any order on the vector. In some embodiments, thenucleotide sequence encoding the Cas9 protein and the nucleotidesequence encoding the guide RNA may be located on the same vector. Insome embodiments, the nucleotide sequence encoding the Cas9 protein andthe template may be located on the same vector. In a particularembodiment, the vector system may comprise a first vector comprising thenucleotide sequence encoding the Cas9 protein, and a second vectorcomprising the nucleotide sequence encoding the template or multiplecopies of the template.

In some embodiments, the template may be released from the vector onwhich it is located by the nuclease system encoded by the vector system.In some embodiments, the template may be released from the vector by aCas9 protein provided from an mRNA. The template may comprise at leastone target sequence that is recognized by the guide RNA. In someembodiments, the template may be flanked by a target sequence at the 5′and 3′ ends of the template. Upon expression of Cas9 protein anddelivery of the guide RNA, the guide RNA may hybridize with and the Cas9protein may cleave the target sequence at both ends of the template suchthat the template is released from the vector. In additionalembodiments, the template may be released from the vector by a nucleaseencoded by the vector system by having a target sequence recognized bythe nuclease at the 5′ and 3′ ends of the template. The target sequencesat either end of the template may be oriented such that the PAM sequenceis closer to the template. In such an orientation, fewer non-templatenucleic acids remain on the ends of the template after release from thevector. In some embodiments, the target sequences flanking the templatemay be the same. In some embodiments, the target sequences flanking thetemplate may be the same as the target sequence found at the cleavagesite in which the template is incorporated, e.g., by HR, HDR, ornon-homologous end joining. In other embodiments, the target sequencesflanking the template may be different. For example, the target sequenceat the 5′ end of the template may be recognized by one guide RNA ornuclease, and the target sequence at the 3′ end of the template may berecognized by another guide RNA or nuclease.

In some embodiments, the vector encoding the nuclease system maycomprise at least one target sequence within the vector, to create aself-destroying (or “self-cleaving” or “self-inactivating”) vectorsystem to control the amount of the nuclease system to be expressed. Insome embodiments, the self-destroying vector system results in areduction in the amount of nuclease activity. In further embodiments,the self-destroying vector system results in a reduction in the amountof vector nucleic acid. In embodiments in which the system comprisesCas9, it also comprises guide RNA(s) that recognize the target sequence.In this way, the residence time and/or the level of activity of thenuclease system may be temporally controlled to avoid adverse effectsassociated with overexpression of the nuclease system. Such adverseeffects may include, e.g., an off-target effect by the nuclease. In someembodiments, one or more target sequences may be located at any place onthe vector such that, upon expression of the nuclease, the nucleaserecognizes and cleaves the target sequence in the vector that containsthe nuclease-encoding sequence. The one or more target sequences of theself-destroying vector may be the same. Optionally, the self-destroyingvector may comprise multiple target sequences. In some embodiments, thecleavage at a target sequence may reduce the expression of at least onecomponent of the nuclease system, such as, for example, Cas9. In someembodiments, the cleavage may reduce the expression of the nucleasetranscript. For example, a target sequence may be located within thenucleotide sequence encoding the nuclease such that the cleavage resultsin the disruption of the coding region. In other embodiments, a targetsequence may be located within a non-coding region on the vectorencoding the nuclease. In some embodiments, a target sequence may belocated within the promoter that drives the expression of the nucleasesuch that the cleavage results in the disruption of the promotersequence. For example, the vector may contain a target sequence (and itscorresponding guide RNA) that targets a Cas9 sequence. In certainembodiments, a target sequence may be located between the promoter andthe nucleotide sequence encoding the nuclease such that the cleavageresults in the separation of the coding sequence from its promoter. Incertain embodiments, a target sequence outside the nuclease codingsequence and a target sequence within the nuclease coding sequence areincluded.

In some embodiments, the vector encoding a Cas9 protein may comprise atleast one target sequence that is recognized by a guide RNA. In someembodiments, the target sequence may be located at any place on thevector such that, upon expression of the Cas9 protein and the guide RNA,the guide RNA hybridizes with and the Cas9 protein cleaves the targetsequence in the vector encoding the Cas9 protein. In some embodiments,the cleavage at the target sequence may reduce the expression of theCas9 protein transcript. For example, the target sequence may be locatedwithin the nucleotide sequence encoding the Cas9 protein such that thecleavage results in the disruption of the coding region. In otherembodiments, the target sequence may be located within a non-codingregion on the vector encoding the Cas9 protein. In some embodiments, thetarget sequence may be located within the promoter that drives theexpression of the Cas9 protein such that the cleavage results in thedisruption of the promoter sequence. In some embodiments, the targetsequence may be located within the nucleotide sequence encoding the Cas9protein such that the cleavage results in the disruption of the codingsequence. In other embodiments, the target sequence may be locatedbetween the promoter and the nucleotide sequence encoding the Cas9protein such that the cleavage results in the separation of the codingsequence from its promoter.

The target sequences for release of the template, for vectorself-destruction, and for targeting by the nuclease system in a cell maybe the same or different. For example, the target sequence at the 3′ endof the template may be present within the promoter driving theexpression of the nuclease (e.g., the Cas9 protein) such that therelease of the template simultaneously results in the disruption of theexpression of the nuclease (e.g., the Cas9 protein). In someembodiments, both target sequences flanking the template, the targetsequences for disrupting the expression of the nuclease (e.g., the Cas9protein), and the target sequence in the target nucleic acid molecule ina cell may be the same sequence that is recognized by a single guide RNAor nuclease. Thus, in some embodiments, the vector system may compriseonly one type of target sequence, and the nuclease system may compriseonly one guide RNA. In other embodiments, these target sequences maycomprise different sequences that are recognized by different guideRNAs.

In some embodiments, the vector system may comprise inducible promotersto start expression only after it is delivered to a target cell.Non-limiting exemplary inducible promoters include those inducible byheat shock, light, chemicals, peptides, metals, steroids, antibiotics,or alcohol. In some embodiments, the inducible promoter may be one thathas a low basal (non-induced) expression level, such as, e.g., theTet-On® promoter (Clontech).

In additional embodiments, the vector system may comprisetissue-specific promoters to start expression only after it is deliveredinto a specific tissue. Non-limiting exemplary tissue-specific promotersinclude albumin promoter, α-1 antitrypsin promoter, hemopexin promoter,B29 promoter, CD14 promoter, CD43 promoter, CD45 promoter, CD68promoter, desmin promoter, elastase-1 promoter, endoglin promoter,fibronectin promoter, Flt-1 promoter, GFAP promoter, GPIIb promoter,ICAM-2 promoter, INF-β promoter, Mb promoter, Nphsl promoter, OG-2promoter, SP-B promoter, SYN1 promoter, and WASP promoter. In particularembodiments, the tissue specific promoter is an albumin promoter, a α-1antitrypsin promoter, a hepatitis B virus core promoter, or a hemopexingene promoter. Methods of examining liver specific promoters aredescribed in Kramer et al., Molecular Therapy 7(3): 375-385 (2003),which is incorporated herein in its entirety by reference.

In some embodiments of the present disclosure, the activity of thenuclease system may be temporally regulated by adjusting the residencetime, the amount, and/or the activity of the expressed components of thenuclease system. For example, as described herein, the nuclease may befused with a protein domain that is capable of modifying theintracellular half-life of the nuclease. In certain embodimentsinvolving two or more vectors (e.g., a vector system in which thecomponents described herein are encoded on two or more separatevectors), the activity of the nuclease system may be temporallyregulated by controlling the timing in which the vectors are delivered.For example, in some embodiments a vector encoding the nuclease systemmay deliver the nuclease prior to the vector encoding the template. Inother embodiments, the vector encoding the template may deliver thetemplate prior to the vector encoding the nuclease system. In someembodiments, the vectors encoding the nuclease system and template aredelivered simultaneously. In certain embodiments, the simultaneouslydelivered vectors temporally deliver, e.g., the nuclease, template,and/or guide RNA components. In further embodiments, the RNA (such as,e.g., the nuclease transcript) transcribed from the coding sequence onthe vectors may further comprise at least one element that is capable ofmodifying the intracellular half-life of the RNA and/or modulatingtranslational control. In some embodiments, the half-life of the RNA maybe increased. In some embodiments, the half-life of the RNA may bedecreased. In some embodiments, the element may be capable of increasingthe stability of the RNA. In some embodiments, the element may becapable of decreasing the stability of the RNA. In some embodiments, theelement may be within the 3′ UTR of the RNA. In some embodiments, theelement may include a polyadenylation signal (PA). In some embodiments,the element may include a cap, e.g., an upstream mRNA end. In someembodiments, the PA may be added to the 3′ UTR of the RNA. In someembodiments, the RNA may comprise no PA such that it is subject toquicker degradation in the cell after transcription. In someembodiments, the element may include at least one AU-rich element (ARE).The AREs may be bound by ARE binding proteins (ARE-BPs) in a manner thatis dependent upon tissue type, cell type, timing, cellular localization,and environment. In some embodiments the destabilizing element maypromote RNA decay, affect RNA stability, or activate translation. Insome embodiments, the ARE may comprise 50 to 150 nucleotides in length.In some embodiments, the ARE may comprise at least one copy of thesequence AUUUA. In some embodiments, at least one ARE may be added tothe 3′ UTR of the RNA. In some embodiments, the element may be aWoodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element(WPRE), which creates a tertiary structure to enhance expression fromthe transcript. In further embodiments, the element is a modified and/ortruncated WPRE sequence that is capable of enhancing expression from thetranscript, as described, for example in Zufferey et al., J Virol,73(4): 2886-92 (1999) and Flajolet et al., J Virol, 72(7): 6175-80(1998). In some embodiments, the WPRE or equivalent may be added to the3′ UTR of the RNA. In some embodiments, the element may be selected fromother RNA sequence motifs that are enriched in either fast- orslow-decaying transcripts.

Embodiments of the disclosure also encompass treating a patient with thevector system described herein. In some embodiments, the method maycomprise administering the vector system described herein to thepatient. The method may be used as a single therapy or in combinationwith other therapies available in the art. In some embodiments, thepatient may have a mutation (such as, e.g., insertion, deletion,substitution, chromosome translocation) in a disease-associated gene. Insome embodiments, administration of the vector system may result in amutation comprising an insertion, deletion, or substitution of one ormore nucleotides of the disease-associated gene in the patient. Certainembodiments may include methods of repairing the patient's mutation inthe disease-associated gene. In some embodiments, the mutation mayresult in one or more amino acid changes in a protein expressed from thedisease-associated gene. In some embodiments, the mutation may result inone or more nucleotide changes in an RNA expressed from thedisease-associated gene. In some embodiments, the mutation may alter theexpression level of the disease-associated gene. In some embodiments,the mutation may result in increased or decreased expression of thegene. In some embodiments, the mutation may result in gene knockdown inthe patient. In some embodiments, the administration of the vectorsystem may result in the correction of the patient's mutation in thedisease-associated gene. In some embodiments, the administration of thevector system may result in gene knockout in the patient. In someembodiments, the administration of the vector system may result inreplacement of an exon sequence, an intron sequence, a transcriptionalcontrol sequence, a translational control sequence, or a non-codingsequence of the disease-associated gene.

In some embodiments, the administration of the vector system may resultin integration of an exogenous sequence of the template into thepatient's genomic DNA. In some embodiments, the exogenous sequence maycomprise a protein or RNA coding sequence operably linked to anexogenous promoter sequence such that, upon integration of the exogenoussequence into the patient's genomic DNA, the patient is capable ofexpressing the protein or RNA encoded by the integrated sequence. Theexogenous sequence may provide a supplemental or replacement proteincoding or non-coding sequence. For example, the administration of thevector system may result in the replacement of the mutant portion of thedisease-associated gene in the patient. In some embodiments, the mutantportion may include an exon of the disease-associated gene. In otherembodiments, the integration of the exogenous sequence may result in theexpression of the integrated sequence from an endogenous promotersequence present on the patient's genomic DNA. For example, theadministration of the vector system may result in supply of a functionalgene product of the disease-associated gene to rectify the patient'smutation. In some embodiments, the administration of the vector systemmay result in integration of a cDNA sequence encoding a protein or aportion of the protein. In yet other embodiments, the administration ofthe vector system may result in integration of an exon sequence, anintron sequence, a transcriptional control sequence, a translationalcontrol sequence, or a non-coding sequence into the patient's genomicDNA. In some embodiments, the administration of the vector system mayresult in gene knockin in the patient.

Administration and Method of Use

Provided herein are methods and compositions for editing a targetnucleic acid in a cell. Further provided herein are pharmaceuticalcompositions and methods for modifying the function and activity of atarget gene in a cell of a subject. The genome editing compositionsdescribed herein may be administered to a subject in need thereof, in atherapeutically effective amount, to treat conditions related to highcirculating cholesterol levels and/or coronary disease, e.g.hypercholesterolemia, elevated total cholesterol levels, elevatedlow-density lipoprotein (LDL) levels, elevated LDL-cholesterol levels,reduced high-density lipoprotein levels, liver steatosis, coronary heartdisease, ischemia, stroke, peripheral vascular disease, thrombosis, type2 diabetes, high elevated blood pressure, atherosclerosis, obesity,Alzheimer's disease, neurodegeneration, and combinations thereof can beadministered to the subject in a variety of ways, includingparenterally, intravenously, intradermally, intramuscularly,colonically, rectally or intraperitoneally. In some embodiments, thepharmaceutical composition may be co-administered with pharmaceuticallyacceptable salt by intraperitoneal injection, intramuscular injection,subcutaneous injection, or intravenous injection of the subject. In someembodiments, the pharmaceutical composition may be directly injected toa specific tissue, such as the liver tissue. In some embodiments, thepharmaceutical compositions can be administered parenterally,intravenously, intramuscularly or orally. The oral formulations can befurther coated or treated to prevent or reduce dissolution in stomach.The compositions of the present disclosure can be administered to asubject using any suitable methods known in the art. Suitableformulations for use in the present disclosure and methods of deliveryare generally well known in the art. For example, the composition of thepresent disclosure can be formulated as pharmaceutical compositions witha pharmaceutically acceptable diluent, carrier or excipient. Thecompositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions includingpH adjusting and buffering agents, tonicity adjusting agents, wettingagents and the like, such as, for example, sodium acetate, sodiumlactate, sodium chloride, potassium chloride, calcium chloride, sorbitanmonolaurate, triethanolamine oleate, etc.

Pharmaceutical formulations described herein can be administrable to asubject in a variety of ways by multiple administration routes,including but not limited to, oral, parenteral (e.g., intravenous,subcutaneous, intramuscular, intramedullary injections, intrathecal,direct intraventricular, intraperitoneal, intralymphatic, intranasalinjections), intranasal, buccal, topical or transdermal administrationroutes. The pharmaceutical formulations described herein include, butare not limited to, aqueous liquid dispersions, self-emulsifyingdispersions, solid solutions, liposomal dispersions, aerosols, soliddosage forms, powders, immediate release formulations, controlledrelease formulations, fast melt formulations, tablets, capsules, pills,delayed release formulations, extended release formulations, pulsatilerelease formulations, multiparticulate formulations, and mixed immediateand controlled release formulations.

In some embodiments, the pharmaceutical formulation is in the form of atablet. In other embodiments, pharmaceutical formulations containing ancomposition or inhibitory agent described herein are in the form of acapsule. In one aspect, liquid formulation dosage forms for oraladministration are in the form of aqueous suspensions or solutionsselected from the group including, but not limited to, aqueous oraldispersions, emulsions, solutions, elixirs, gels, and syrups.

For administration by inhalation, a composition or inhibitory agentdescribed herein can be formulated for use as an aerosol, a mist or apowder. For buccal or sublingual administration, the compositions maytake the form of tablets, lozenges, or gels formulated in a conventionalmanner. In some embodiments, a composition or inhibitory agent describedherein can be prepared as transdermal dosage forms. In some embodiments,a composition or inhibitory agent described herein can be formulatedinto a pharmaceutical composition suitable for intramuscular,subcutaneous, or intravenous injection. In some embodiments, acomposition or inhibitory agent described herein can be administeredtopically and can be formulated into a variety of topicallyadministrable compositions, such as solutions, suspensions, lotions,gels, pastes, medicated sticks, balms, creams or ointments. In someembodiments, a composition or inhibitory agent described herein can beformulated in rectal compositions such as enemas, rectal gels, rectalfoams, rectal aerosols, suppositories, jelly suppositories, or retentionenemas.

In one aspect, disclosed herein is a method of treating a disease orcondition in a mammal, the method comprising administering to a mammal atherapeutically effective amount of a herein described pharmaceuticalcomposition. In one aspect, disclosed herein are methods for treating adisease or condition, including raising an immune response to animmunogen, in a subject. In one embodiment, the disease or condition istreatable by administering the payload. In some embodiments, the diseaseor condition is characterized by missing or aberrant protein orpolypeptide activity. For example, an LNP composition comprising an mRNAencoding a missing or aberrant polypeptide may be administered ordelivered to a cell. Subsequent translation of the mRNA may produce thepolypeptide, thereby reducing or eliminating an issue caused by theabsence of or aberrant activity caused by the polypeptide. A payloadincluded in an LNP composition may also be capable of altering the rateof transcription of a given species, thereby affecting gene expression.

Diseases and/or conditions characterized by dysfunctional or aberrantprotein or polypeptide activity can include, but are not limited to,rare diseases, infectious diseases (as both vaccines and therapeutics),cancer and proliferative diseases, genetic diseases (e.g., cysticfibrosis), autoimmune diseases, diabetes, neurodegenerative diseases,cardio- and reno-vascular diseases, and metabolic diseases. Multiplediseases and/or conditions may be characterized by missing (orsubstantially diminished such that proper protein function does notoccur) protein activity. Such proteins may not be present, or they maybe essentially non-functional. A specific example of a dysfunctionalprotein is the missense mutation variants of the cystic fibrosistransmembrane conductance regulator (CFTR) gene. In some embodiments,the present disclosure provides a method for treating such diseasesand/or conditions in a subject by administering an LNP composition orpharmaceutical composition comprising an RNA payload, wherein the RNAcan be an mRNA encoding a polypeptide that antagonizes or otherwiseovercomes an aberrant protein activity present in the cell of thesubject.

Dosage

Appropriate dosage or effective amounts for administration vary, asrecognized by those skilled in the art, depending on the particularcondition being treated, the severity of the condition, the individualsubject parameters including age, physical condition, size, gender andweight, the duration of the treatment, the nature of concurrent therapy(if any), the specific route of administration and like factors withinthe knowledge and expertise of the health practitioner. Factors involvedin dosage determination are known to those of ordinary skill in the artwithout additional experimentation other than routine test. It isgenerally preferred that a maximum dose of the individual components orcombinations thereof be used, that is, the highest safe dose accordingto sound medical judgment. Empirical considerations, such as thehalf-life, generally will contribute to the determination of the dosage.For example, therapeutic agents that are compatible with the humanimmune system, such as polypeptides comprising regions from humanizedantibodies or fully human antibodies, may be used to prolong half-lifeof the polypeptide and to prevent the polypeptide being attacked by thehost's immune system.

Frequency of administration may be determined and adjusted over thecourse of therapy, and is generally, but not necessarily, based ontreatment and/or suppression and/or amelioration and/or delay of adisease. Alternatively, sustained continuous release formulations of apolypeptide or a polynucleotide may be appropriate. Various formulationsand devices for achieving sustained release are known in the art. Insome embodiments, dosage is daily, every other day, every three days,every four days, every five days, or every six days. In someembodiments, dosing frequency is once every week, every 2 weeks, every 4weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every9 weeks, or every 10 weeks; or once every month, every 2 months, orevery 3 months, or longer. The progress of this therapy is easilymonitored by conventional techniques and assays.

The dosing regimen can vary over time. In some embodiments, for an adultsubject of normal weight, doses ranging from about 0.01 to 1000 mg/kgmay be administered. In some embodiments, the dose is between 1 to 200mg. In some embodiments, the doses may range from about 0.01 to 0.05mg/kg, between about 0.01 to 0.1 mg/kg, between about 0.01 to 1 mg/kg,between about 0.01 to 10 mg/kg, between about 0.01 to 100 mg/kg, between0.01 to 500 mg/kg, between about 0.1 to 1 mg/kg, between about 0.1 to 5mg/kg, between about 0.1 to 10 mg/kg, between about 0.1 to 100 mg/kg,between about 0.1 to 500 mg/kg, between about 0.1 to 1000 mg/kg, betweenabout 1 to 5 mg/kg, between about 1 to 10 mg/kg, between about 1 to 100mg/kg, between about 1 to 500 mg/kg, between about 1 to 1000 mg/kg,between about 10 to 100 mg/kg, between about 10 to 500 mg/kg, betweenabout 10 to 1000 mg/kg, or between about 100 to 1000 mg/kg. Theparticular dosage regimen, i.e., dose, timing and repetition, willdepend on the particular subject and that subject's medical history, aswell as the properties of the polypeptide or the polynucleotide (such asthe half-life of the polypeptide or the polynucleotide, and otherconsiderations well known in the art).

As will be apparent to those skilled in the art, the appropriate dosageof a therapeutic agent as described herein will depend on the specificagent (or compositions thereof) employed, the formulation and route ofadministration, the type and severity of the disease, whether thepolypeptide or the polynucleotide is administered for preventive ortherapeutic purposes, previous therapy, the subject's clinical historyand response to the antagonist, and the discretion of the attendingphysician. Typically the clinician will administer a polypeptide until adosage is reached that achieves the desired result.

Administration of one or more therapeutic compositions, e.g.polypeptides, polynucleotides, or RNPs, can be continuous orintermittent, depending, for example, upon the recipient's physiologicalcondition, whether the purpose of the administration is therapeutic orprophylactic, and other factors known to skilled practitioners. Theadministration of a polypeptide may be essentially continuous over apreselected period of time or may be in a series of spaced dose, e.g.,either before, during, or after developing a disease.

Biological Samples

A sample, e.g., a biological sample can be taken from a subject. Abiological sample can comprise a plurality of biological samples. Theplurality of biological samples can contain two or more biologicalsamples; for examples, about 2-1000, 2-500, 2-250, 2-100, 2-75, 2-50,2-25, 2-10, 10-1000, 10-500, 10-250, 10-100, 10-75, 10-50, 10-25,25-1000, 25-500, 25-250, 25-100, 25-75, 25-50, 50-1000, 50-500, 50-250,50-100, 50-75, 60-70, 100-1000, 100-500, 100-250, 250-1000, 250-500,500-1000, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 90, 95, 100, 110, 120, 130,140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300,325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800,850, 900, 950, 1000, or more biological samples. The biological samplescan be obtained from a plurality of subjects, giving a plurality of setsof a plurality of samples. The biological samples can be obtained fromabout 2 to about 1000 subjects, or more; for example, about 2-1000,2-500, 2-250, 2-100, 2-50, 2-25, 2-20, 2-10, 10-1000, 10-500, 10-250,10-100, 10-50, 10-25, 10-20, 15-20, 25-1000, 25-500, 25-250, 25-100,25-50, 50-1000, 50-500, 50-250, 50-100, 100-1000, 100-500, 100-250,250-1000, 250-500, 500-1000, or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45,50, 55, 60, 65, 68, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325,350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850,900, 950, or 1000 or more subjects.

The biological samples can be obtained from human subjects. Thebiological samples can be obtained from human subjects at differentages. The human subject can be prenatal (e.g., a fetus), a child (e.g.,a neonate, an infant, a toddler, a preadolescent), an adolescent, apubescent, or an adult (e.g., an early adult, a middle aged adult, asenior citizen). The human subject can be between about 0 months andabout 120 years old, or older. The human subject can be between about 0and about 12 months old; for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, or 12 months old. The human subject can be between about 0 and12 years old; for example, between about 0 and 30 days old; betweenabout 1 month and 12 months old; between about 1 year and 3 years old;between about 4 years and 5 years old; between about 4 years and 12years old; about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years old. Thehuman subject can be between about 13 years and 19 years old; forexample, about 13, 14, 15, 16, 17, 18, or 19 years old. The humansubject can be between about 20 and about 39 year old; for example,about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, or 39 years old. The human subject can be between about 40to about 59 years old; for example, about 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59 years old. Thehuman subject can be greater than 59 years old; for example, about 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,112, 113, 114, 115, 116, 117, 118, 119, or 120 years old. The humansubjects can include living subjects or deceased subjects. The humansubjects can include male subjects and/or female subjects.

Biological samples can be obtained from any suitable source that allowsdetermination of expression levels of genes, e.g., from cells, tissues,bodily fluids or secretions, or a gene expression product derivedtherefrom (e.g., nucleic acids, such as DNA or RNA; polypeptides, suchas protein or protein fragments). The nature of the biological samplecan depend upon the nature of the subject. If a biological sample isfrom a subject that is a unicellular organism or a multicellularorganism with undifferentiated tissue, the biological sample cancomprise cells, such as a sample of a cell culture, an excision of theorganism, or the entire organism. If a biological sample is from amulticellular organism, the biological sample can be a tissue sample, afluid sample, or a secretion.

The biological samples can be obtained from different tissues. The termtissue is meant to include ensembles of cells that are of a commondevelopmental origin and have similar or identical function. The termtissue is also meant to encompass organs, which can be a functionalgrouping and organization of cells that can have different origins. Thebiological sample can be obtained from any tissue.

The biological samples can be obtained from different tissue samplesfrom one or more humans or non-human animals. Suitable tissues caninclude connective tissues, muscle tissues, nervous tissues, epithelialtissues or a portion or combination thereof. Suitable tissues can alsoinclude all or a portion of a lung, a heart, a blood vessel (e.g.,artery, vein, capillary), a salivary gland, a esophagus, a stomach, aliver, a gallbladder, a pancreas, a colon, a rectum, an anus, ahypothalamus, a pituitary gland, a pineal gland, a thyroid, aparathyroid, an adrenal gland, a kidney, a ureter, a bladder, a urethra,a lymph node, a tonsil, an adenoid, a thymus, a spleen, skin, muscle, abrain, a spinal cord, a nerve, an ovary, a fallopian tube, a uterus,vaginal tissue, a mammary gland, a testicle, a vas deferens, a seminalvesicle, a prostate, penile tissue, a pharynx, a larynx, a trachea, abronchi, a diaphragm, bone marrow, a hair follicle, or a combinationthereof. A biological sample from a human or non-human animal can alsoinclude a bodily fluid, secretion, or excretion; for example, abiological sample can be a sample of aqueous humour, vitreous humour,bile, blood, blood serum, breast milk, cerebrospinal fluid, endolymph,perilymph, female ejaculate, amniotic fluid, gastric juice, menses,mucus, peritoneal fluid, pleural fluid, saliva, sebum, semen, sweat,tears, vaginal secretion, vomit, urine, feces, or a combination thereof.The biological sample can be from healthy tissue, diseased tissue,tissue suspected of being diseased, or a combination thereof.

In some embodiments, the biological sample is a fluid sample, forexample a sample of blood, serum, sputum, urine, semen, or otherbiological fluid. In certain embodiments the sample is a blood sample.In some embodiments the biological sample is a tissue sample, such as atissue sample taken to determine the presence or absence of disease inthe tissue. In certain embodiments the sample is a sample of thyroidtissue.

The biological samples can be obtained from subjects in different stagesof disease progression or different conditions. Different stages ofdisease progression or different conditions can include healthy, at theonset of primary symptom, at the onset of secondary symptom, at theonset of tertiary symptom, during the course of primary symptom, duringthe course of secondary symptom, during the course of tertiary symptom,at the end of the primary symptom, at the end of the secondary symptom,at the end of tertiary symptom, after the end of the primary symptom,after the end of the secondary symptom, after the end of the tertiarysymptom, or a combination thereof. Different stages of diseaseprogression can be a period of time after being diagnosed or suspectedto have a disease; for example, at least about, or at least, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or24 hours; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 days; 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 weeks; 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11 or 12 months; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49or 50 years after being diagnosed or suspected to have a disease.Different stages of disease progression or different conditions caninclude before, during or after an action or state; for example,treatment with drugs, treatment with a surgery, treatment with aprocedure, performance of a standard of care procedure, resting,sleeping, eating, fasting, walking, running, performing a cognitivetask, sexual activity, thinking, jumping, urinating, relaxing, beingimmobilized, being emotionally traumatized, being shock, and the like.

The methods of the present disclosure provide for analysis of abiological sample from a subject or a set of subjects. The subject(s)may be, e.g., any animal (e.g., a mammal), including but not limited tohumans, non-human primates, rodents, dogs, cats, pigs, fish, and thelike. The present methods and compositions can apply to biologicalsamples from humans, as described herein.

A biological sample can be obtained by methods known in the art such asthe biopsy methods provided herein, swabbing, scraping, phlebotomy, orany other suitable method. The biological sample can be obtained,stored, or transported using components of a kit of the presentdisclosure. In some cases, multiple biological samples, such as multiplethyroid samples, can be obtained for analysis, characterization, ordiagnosis according to the methods of the present disclosure. In somecases, multiple biological samples, such as one or more samples from onetissue type (e.g., thyroid) and one or more samples from another tissuetype (e.g., buccal) can be obtained for diagnosis or characterization bythe methods of the present disclosure. In some cases, multiple samples,such as one or more samples from one tissue type (e.g., thyroid) and oneor more samples from another tissue (e.g., buccal) can be obtained atthe same or different times. In some cases, the samples obtained atdifferent times are stored and/or analyzed by different methods. Forexample, a sample can be obtained and analyzed by cytological analysis(e.g., using routine staining). In some cases, a further sample can beobtained from a subject based on the results of a cytological analysis.The diagnosis of a disease or condition, e.g. a coronary disease caninclude examination of a subject by a physician, nurse or other medicalprofessional. The examination can be part of a routine examination, orthe examination can be due to a specific complaint including, but notlimited to, one of the following: pain, illness, anticipation ofillness, presence of a suspicious lump or mass, a disease, or acondition. The subject may or may not be aware of the disease orcondition. The medical professional can obtain a biological sample fortesting. In some cases the medical professional can refer the subject toa testing center or laboratory for submission of the biological sample.The methods of obtaining provided herein include methods of biopsyincluding fine needle aspiration, core needle biopsy, vacuum assistedbiopsy, incisional biopsy, excisional biopsy, punch biopsy, shave biopsyor skin biopsy. In some cases, the methods and compositions providedherein are applied to data only from biological samples obtained by FNA.In some cases, the methods and compositions provided herein are appliedto data only from biological samples obtained by FNA or surgical biopsy.In some cases, the methods and compositions provided herein are appliedto data only from biological samples obtained by surgical biopsy. Abiological sample can be obtained by non-invasive methods, such methodsincluding, but not limited to: scraping of the skin or cervix, swabbingof the cheek, saliva collection, urine collection, feces collection,collection of menses, tears, or semen. The biological sample can beobtained by an invasive procedure, such procedures including, but notlimited to: biopsy, alveolar or pulmonary lavage, needle aspiration, orphlebotomy. The method of biopsy can further include incisional biopsy,excisional biopsy, punch biopsy, shave biopsy, or skin biopsy. Themethod of needle aspiration can further include fine needle aspiration,core needle biopsy, vacuum assisted biopsy, or large core biopsy.Multiple biological samples can be obtained by the methods herein toensure a sufficient amount of biological material. Generic methods forobtaining biological samples are also known in the art and furtherdescribed in for example Ramzy, Ibrahim Clinical Cytopathology andAspiration Biopsy 2001 which is herein incorporated by reference in itsentirety. The biological sample can be a fine needle aspirate of athyroid nodule or a suspected thyroid tumor. The fine needle aspiratesampling procedure can be guided by the use of an ultrasound, X-ray, orother imaging device.

In some cases, the subject can be referred to a specialist such as anoncologist, surgeon, or endocrinologist for further diagnosis. Thespecialist can likewise obtain a biological sample for testing or referthe individual to a testing center or laboratory for submission of thebiological sample. In any case, the biological sample can be obtained bya physician, nurse, or other medical professional such as a medicaltechnician, endocrinologist, cytologist, phlebotomist, radiologist, or apulmonologist. The medical professional can indicate the appropriatetest or assay to perform on the sample, or the molecular profilingbusiness of the present disclosure can consult on which assays or testsare most appropriately indicated. The molecular profiling business canbill the individual or medical or insurance provider thereof forconsulting work, for sample acquisition and or storage, for materials,or for all products and services rendered.

A medical professional need not be involved in the initial diagnosis orsample acquisition. An individual can alternatively obtain a samplethrough the use of an over the counter kit. The kit can contain a meansfor obtaining said sample as described herein, a means for storing thesample for inspection, and instructions for proper use of the kit. Insome cases, molecular profiling services are included in the price forpurchase of the kit. In other cases, the molecular profiling servicesare billed separately.

A biological sample suitable for use by the molecular profiling businesscan be any material containing tissues, cells, nucleic acids, genes,gene fragments, expression products, gene expression products, and/orgene expression product fragments of an individual to be tested. Methodsfor determining sample suitability and/or adequacy are provided. Thebiological sample can include, but is not limited to, tissue, cells,and/or biological material from cells or derived from cells of anindividual. The sample can be a heterogeneous or homogeneous populationof cells or tissues. The biological sample can be obtained using anymethod known to the art that can provide a sample suitable for theanalytical methods described herein.

Obtaining a biological sample can be aided by the use of a kit. A kitcan be provided containing materials for obtaining, storing, and/orshipping biological samples. The kit can contain, for example, materialsand/or instruments for the collection of the biological sample (e.g.,sterile swabs, sterile cotton, disinfectant, needles, syringes,scalpels, anesthetic swabs, knives, curette blade, liquid nitrogen,etc.). The kit can contain, for example, materials and/or instrumentsfor the storage and/or preservation of biological samples (e.g.,containers; materials for temperature control such as ice, ice packs,cold packs, dry ice, liquid nitrogen; chemical preservatives or bufferssuch as formaldehyde, formalin, paraformaldehyde, glutaraldehyde,alcohols such as ethanol or methanol, acetone, acetic acid, HOPEfixative (Hepes-glutamic acid buffer-mediated organic solvent protectioneffect), heparin, saline, phosphate buffered saline, TAPS, bicine, Tris,tricine, TAPSO, HEPES, TES, MOPS, PIPES, cadodylate, SSC, IVIES,phosphate buffer; protease inhibitors such as aprotinin, bestatin,calpain inhibitor I and II, chymostatin, E-64, leupeptin,alpha-2-macroglobulin, pefabloc SC, pepstatin, phenylmethanesufonylfluoride, trypsin inhibitors; DNAse inhibitors such as2-mercaptoethanol, 2-nitro-5-thicyanobenzoic acid, calcium, EGTA, EDTA,sodium dodecyl sulfate, iodoacetate, etc.; RNAse inhibitors such asribonuclease inhibitor protein; double-distilled water; DEPC(diethyprocarbonate) treated water, etc.). The kit can containinstructions for use. The kit can be provided as, or contain, a suitablecontainer for shipping. The shipping container can be an insulatedcontainer. The shipping container can be self-addressed to a collectionagent (e.g., laboratory, medical center, genetic testing company, etc.).The kit can be provided to a subject for home use or use by a medicalprofessional. Alternatively, the kit can be provided directly to amedical professional.

One or more biological samples can be obtained from a given subject. Insome cases, between about 1 and about 50 biological samples are obtainedfrom the given subject; for example, about 1-50, 1-40, 1-30, 1-25, 1-20,1-15, 1-10, 1-7, 1-5, 5-50, 5-40, 5-30, 5-25, 5-15, 5-10, 10-50, 10-40,10-25, 10-20, 25-50, 25-40, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, or 50 biological samples can be obtained from the givensubject. Multiple biological samples from the given subject can beobtained from the same source (e.g., the same tissue), e.g., multipleblood samples, or multiple tissue samples, or from multiple sources(e.g., multiple tissues). Multiple biological samples from the givensubject can be obtained at the same time or at different times. Multiplebiological samples from the given subject can be obtained at the samecondition or different condition. Multiple biological samples from thegiven subject can be obtained at the same disease progression ordifferent disease progression of the subject. If multiple biologicalsamples are collected from the same source (e.g., the same tissue) fromthe particular subject, the samples can be combined into a singlesample. Combining samples in this way can ensure that enough material isobtained for testing and/or analysis.

Provided herein are methods and compositions for targeted delivery oftherapeutic agents such as guide RNAs or guide RNA-Cas complexes. Thepresent inventors have surprisingly found that distinct structures ofGalNAc and GalNAc derivative targeting moieties conjugated with guideRNA display high tissue specific delivery efficiency, and maintains theability to bind and modify target DNA. Advantageously, modified guideRNAs covalently conjugated with GalNAc targeting moiety, as well asguide RNAs connected to GalNAc targeting moiety through nucleic acidbase pairing and hybridization show stability and effective specificdelivery to liver. The inventors show for the first time thatconjugation of gRNA with distinct GalNAc moieties, either by covalentlinkage or by hybridization efficiently directs the gRNA or gRNA-Cas9complex to hepatocytes, and maintain sgRNA integrity, secondarystructure stability, as well as CRISPR enzyme activity and increasedCRISPR editing efficacy in vivo.

EXAMPLES

The following examples are provided to better illustrate the presentdisclosure and are not to be interpreted as limiting the scope of thedisclosure. To the extent that specific materials are mentioned, it ismerely for purposes of illustration and is not intended to limit thedisclosure. One skilled in the art may develop equivalent means orreactants without the exercise of inventive capacity and withoutdeparting from the scope of the disclosure.

Example 1. Synthesis of N-Acetylgalcosamine Derived Monomers forConjugation to Nucleic Acids/Oligonucleotides

Compound 6 is prepared starting from the activated sugar 1 as reported(WO 2018/136620 A2). Compound 5 is purchased from a commercial source.

^(a)Optically pure R, S and racemic versions of all the intermediatesand target compounds are prepared starting from the correspondingchirally pure or racemic starting materials.

Compound 10 are purchased from commercial sources or prepared asreported in the literature (Bull. Chem. Soc. Japan (1998), 71(3),717-721; J. Med. Chem. (2010), 53(1), 432-440, US20120114696 A1). Theamine-protected R, S and racemic lysine 16 is purchased from commercialsources. The fully protected spacer 21 in optically pure and racemicforms are prepared starting from compound 10. The sugar-protectedN-acetylgalactosamine (GalNAc) derivative 25 is prepared according toreported procedure (WO 2018/136620 A2). The intermediate compound 23 isprepared from compound 18 and commercially available N-Boc amino acid22. The fully protected sugar intermediate compound 26 is prepared fromcompound 23 and the sugar intermediate 25.

^(a)Optically pure R, S and racemic versions of all the intermediatesand target compounds are prepared starting from the correspondingchirally pure or racemic starting materials.

Desired lactones 27 are purchased from commercially available sources.Compound 32 is prepared from D-galactosamine as reported (WO 2018/136620A2). The amine intermediate 31 is prepared from compound 10 and desiredlactone 27. Compound 31 is then reacted with the acid 32 under peptidecoupling conditions to obtain compound 33, which is then subjected tohydrogenation over Pd—C to obtain the amine intermediate 34. The aminethen is coupled with the acid 25 to obtain compound 35. Treatmentcompound 35 with HF-py affords compound 36. Phosphitylation of compound36 affords the phosphoramidite 37 (WO 2018/136620 A2). Treatment ofcompound 36 with succinic anhydride in the presence of DMAP followed bytreatment of the semi-succinate with amine-functionalized solid supportunder peptide coupling conditions affords the solid support 38.Unreacted amine on the support are capped by treating with aceticanhydride.

Compound 39, purchased from a commercial source, is treated with lithiumborohydride to obtain the triol 40. Compound 40 upon treatment withmethyl acrylate under Michael addition conditions affords compound 41.Hydrolysis of the triester 41 afforded the tri-acid 42, which is thencoupled with the amine 4 under peptide coupling conditions to affordcompound 43.

Compound 40 is treated with acrylonitrile under Michael additionconditions to obtain compound 44, which is subsequently treated withRaney-Ni under hydrogen to obtain the amine 45. The amine 45 is treatedwith the acid 25 under peptide coupling condition to obtain compound 46.

Compound 49 is prepared from compounds 41 and 30. Treatment of compound41 with acid followed by reaction with desired anhydride 19 affordedacid 46. 1 mol equivalent of acid 46 is treated with one mol equivalentof the amine 30 under peptide coupling conditions to obtain compound 47.Treatment of the amine 47 with Cbz-Cl in the presense of base followedby treatment with LiOH affords compound 49. Reaction of compound 49 withexcess amine 4 under peptide coupling condition yields compound 50.Treatment of compound 50 with hydrogen over Pd—C affords compound 51,which is then coupled with the acid 32 under peptide coupling conditionsaffords compound 52. Treatment of compound 52 with Py-HF yields compound53. Phosphitylation of 53 affords the phosphoramidite 54. Treatment of53 with succinic anhydride in the presence of base followed by treatmentwith amine-functionalized solid support under peptide couplingconditions yields the solid support 55. Unreacted amine on the support55 is capped by treating with acetic anhydride in the presence of abase.

^(a)Optically pure R, S and racemic versions of all the intermediatesand target compounds are prepared starting from the correspondingchirally pure or racemic starting materials.

The acid 62 is prepared from commercially available methyl ester ofhydroxy acid(s) 56. The hydroxyl group of the compound 56 is protectedas DMTr and then the ester is hydrolyzed to obtain the acid 59. Compound59 is reacted with hydrochloride salt of methionine methyl ester underpeptide coupling conditions in the presence of base and then with LiOHin the presence of water to obtain the acid 60. The acid 60 issuccessively reacted with (1) the amine hydrochloride 61 under peptidecoupling conditions to form the amide bond; (2) TBDMS-Cl in the presenceof imidazole and (3) with LiOH in the presence of water to obtaincompound 62. The amine salt 63 is prepared from compound 43. Compound 63is then reacted with the compound 62 under peptide coupling conditionsto yield compound 64. Treatment of 64 with Py-HF affords compound 65.Phosphitylation of 65 yields the phosphoramidite 66. Treatment of 65successively with succinic anhydride in the presence of DMAP and thenwith amine-functionalized solid support under peptide couplingconditions yields the solid support 67. The unreacted amine on the solidsupport was then quenched by reacting with acetic anhydride in thepresence of a base.

The phosphoramidite 68 and the solid support 69 are prepared fromcompound 46 and 62 as shown in the first part of Scheme 6 and asdescribed above.

^(a)Optically pure R, S and racemic versions of all the intermediatesand target compounds are prepared starting from the correspondingchirally pure or racemic starting materials.

The phosphoramidite 73 and the solid support 74 are prepared fromdesired starting material(s) 23 as shown in the Scheme 7. Treatment of23 with acid affords compound 70. Compound 70 is then reacted withcompound 60 under peptide coupling conditions to obtain compound 71.Compound 71 is hydrogenated over Pd—C at atmospheric pressure and thereacted with compound 25 under peptide coupling conditions to compound72. Phosphitylation of compound 72 affords the phosphoramidite 73.Treatment of compound 72 with succinic anhydride in the presence of abase followed treatment with amine-functionalized solid support underpeptide coupling conditions affords the solid support 74. Unreactedamine on the support obtained is quenched by reacting with aceticanhydride in the presence of a base to yield the solid support ready fornucleic acid/oligonucleotide synthesis.

Example 2. GalNAc Conjugate Synthesis

The desired nucleic acid conjugates are synthesized using the solidsupport and phosphoramidites described in Scheme 1-7 and in thepublication Brown et al., NUCLEIC ACID THERAPEUTICS (DOI:10.1089/nat.2019.0782) and as described in the publications: Rajeev, etal., ChemBioChem 2015, 16, 903-908, Nair et al., J. Am. Chem. Soc. 2014,136, 16958-16961 and WO 2018/136620 A2.

Example 3. Targeted Delivery of mRNA to Hepatocytes In Vitro

The RNA poly(A) tail is annealed with short complementaryoligonucleotides conjugated with GalNAc ligand (Table 2). The singlechemical entity thus formed is incubated with ASGPR expressing, rodent,non-human primates and human primary hepatocytes, and/or hepatoma celllines to enable ASGPR-mediated uptake into the cell to elicit expressionof the corresponding protein. The expression of the protein of interestis assayed to check the efficiency of GalNAc-ASGPR mediated delivery ofthe mRNA. Expression of GFP and GFP-Luc mRNA are used as the probes,initially.

Example 4. Targeted Delivery of mRNA to Hepatocytes In Vivo in Rodentsand Non-Human Primates

The RNA poly(A) tail is annealed with short complementaryoligonucleotides conjugated with GalNAc ligand (Table 2). The singlechemical entity thus formed is subcutaneously or intravenouslyadministered to enable ASGPR-mediated uptake of the RNA payload tohepatocytes to elicit protein expression in liver. Expression of theprotein in the livers of treated animals are assayed at regularintervals, after administration.

Example 5. Targeted Delivery of Guide RNA to ASGPR Expressing Cell LinesIn Vitro

The guide RNA (gRNA) conjugated with GalNAc (Table 1) is incubated withASGPR expressing rodent, non-human primates and human primaryhepatocytes, and/or hepatoma cell lines to enable ASGPR-mediated uptakeinto the cell. The mRNA encoding gene editing protein is delivered tothese cell lines by simple transfection using lipofectamine orequivalent transfection agents or by using AAV or AV vectors to expressthe protein.

The gRNA and modified gRNA is annealed with complementary shortoligonucleotide conjugated with GalNAc is incubated with ASGPRexpressing rodent, non-human primates and human primary hepatocytes, andHepG2 cell lines to enable ASGPR-mediated uptake into the cell. Geneediting in hepatocytes and hepatoma cells are assayed after 12 to 36 hpost incubation with the gRNA.

Example 6. Gene Editing in Hepatocytes

The RNA is delivered to ASGPR expressing cell lines using AAV or AVvector and allowed it to express the ribonucleoprotein (RNP). The cellline is then incubated with GalNAc conjugated guide RNA from Table 2 or3 or constituted from Tables 2 and 3. ASGPR-mediated delivery of theGalNAc conjugated guide RNA into the receptor expressing cells. Theguide RNA after uptake into the cell form complex with the expressed RNPto elicit gene editing.

The guide RNA modified with GalNAc from Table 1 is incubated with thecell lines after certain interval namely 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, or 14 days after administering the AAV or AV vector encodingthe RNA, or at a later timepoint beyond 14 days. Differentconcentrations of gRNA-GalNAc are evaluated at different interval postadministration of the gene editor RNA AAV and or AV vector. Gene editingin hepatocytes and hepatoma cells are assayed after 12 to 36 h postincubation with the gRNA.

Example 7. RNA Encoding RNP of Interest is Administered by LNP-MediatedDelivery to Express the Protein in ASGPR Expressing Cell Lines In Vitro

The guide RNA modified with GalNAc from Table 1 is incubated with thecell lines after certain interval namely 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 18, 24 or 36 h after administering the mRNA. Different weightand molar ratio of the mRNA to guide are also evaluated under the sameexperimental conditions. The highest mRNA to guide ratio is 100:1 andthe lowest is 1:100. The ratio in between 100:1 and 1:100 are alsoevaluated. Gene editing in the livers of treated animals are assayedafter 24 to 96 h post incubation with the gRNA and the results arecompared with untreated controls at the same timepoints.

Example 8. Gene Editing in Liver In Vivo in Rodents and in Non-HumanPrimates

The RNA is delivered to livers of rodent and non-human primates usingAAV or AV vector and allowed it to express the ribonucleoprotein (RNP).Subcutaneous (SC) or intravenous (IV) administration of GalNAcconjugated guide RNA from Table 2 or 3, or constituted from Tables 2 and3 enable ASGPR-mediated delivery of the GalNAc conjugated guide RNA intohepatocytes. The guide RNA after uptake into the hepatocytes formcomplex with the expressed RNP to form RNP-guide RNA complex to producegene editing.

The guide RNA modified with GalNAc from Table 1 is subcutaneously orintravenously administered after certain interval namely 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, or 14 days after administering the AAV or AVvector encoding the RNA, or at a later timepoint beyond 14 days.Different concentrations of gRNA-GalNAc are evaluated at differentinterval post administration of the gene editor RNA AAV and or AVvector. Gene editing in the livers of treated animals are assayed after24 to 96 h post incubation with the gRNA and the results are comparedwith untreated controls at the same timepoints.

Example 9. In Vivo Administration

The RNA encoding RNP of interest is administered by LNP-mediateddelivery to rodents and non-human primates by IV infusion over a periodranging from 30 min to 120 min. The infusion time is determined based onthe total dose and/or total dosing volume of the LNP formulation. Incertain LNP dosing, the monkeys are subjected to steroid pretreatment toavoid acute infusion related reactions. The guide RNA modified withGalNAc from Table 1 in saline or equivalent diluent is administeredsubcutaneously to intravenously after certain interval namely 0, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24 or 36 h after administering themRNA. Different weight and molar ratio of the mRNA to guide are alsoevaluated under the same experimental conditions. The highest mRNA toguide ratio is 100:1 and the lowest is 1:100. The ratio in between 100:1and 1:100 are also evaluated. Gene editing in the livers of treatedanimals are assayed after 24 to 96 h post incubation with the gRNA andthe results are compared with untreated controls at the same timepoints.

Example 10. Targeted Delivery of Gene Editor mRNA and Guide RNA (gRNA)to Hepatocytes In Vitro

ASGPR-expressing primary hepatocytes are incubated with gene-editormRNA-GalNAc single chemical entity and guide RNA-GalNAc conjugate toproduce editing of the targeted gene in hepatocytes. The gene-editormRNA-GalNAc single chemical entity and gRNA-GalNAc conjugate are:

-   -   (1) Co-incubated at different mRNA to gRNA ratio ranging from        100:1 to 1:100 by weight and several ratios in between.    -   (2) The gene-editor mRNA-GalNAc single chemical entity is        incubated with the cell lines first and then different ratio of        the gRNA-guide from Table 2 or 3, or constituted from Tables 2        and 3 is incubated with the same cell lines at intervals namely        0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24 or 36 h after        administering the mRNA. Different weight and molar ratio of the        mRNA to guide are also evaluated under the same experimental        conditions. The highest mRNA to guide ratio is 100:1 and the        lowest is 1:100. The ratio in between 100:1 and 1:100 are also        evaluated.

Gene editing in the treated cells are assayed after 24 to 36 h postincubation with the gRNA and the results are compared with untreatedcontrols at the same timepoints.

Example 11. Targeted Delivery of Gene Editor mRNA and Guide RNA (gRNA)to Liver In Vivo in Rodents and in Non-Human Primates

The gene-editor mRNA-GalNAc single chemical entity and gRNA-GalNAcconjugate to produce editing of the targeted gene in the hepatocytes.The gene-editor mRNA-GalNAc single chemical entity and gRNA-GalNAcconjugate are:

-   -   (1) Co-administered subcutaneously or intravenously at different        mRNA to gRNA ratio ranging from 100:1 to 1:100 by weight and        several ratios in between.    -   (2) The gene-editor mRNA-GalNAc single chemical entity is        incubated with the cell lines first and then different ratio of        the gRNA-guide from Table 2 or 3, or constituted from Tables 1        and 2 is incubated with the same cell lines at intervals namely        0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24 or 36 h after        administering the mRNA. Different weight and molar ratio of the        mRNA to guide are also evaluated under the same experimental        conditions. The highest mRNA to guide ratio is 100:1 and the        lowest is 1:100. The ratio in between 100:1 and 1:100 are also        evaluated.

Example 12. RNP-gRNA Complex Preparation and Evaluation to beIncorporated

Gene editing in the livers of treated animals are assayed after 24 to 96h post incubation with the gRNA and the results are compared withuntreated controls at the same timepoints.

Example 13. In Vivo Administration of Single Chemical Entities ofgRNA-GalNAc and mRNA-GalNAc and from Tables 1 and 2 to Rodents andNon-Human Primates

The single chemical entity gRNA-GalNAc and mRNA-GalNAc from Tables 1 and2 is mixed with other components of the nanoparticles prior to dosing.The gRNA and mRNA to be dosed is individually formulated intonanoparticle compositions. Alternatively, the gRNA and mRNA can bepre-mixed before the formation of nanoparticles. After mixing the mRNAand gRNA are dosed and the gene editing in the livers of treated animalsare assayed as described in Example 11.

Example 14. Synthesis of N-Acetylgalcosamine-Lipid (GalNAc-Lipid)Conjugates to Constitute GalNAc-LNPs for Targeted Delivery toHepatocytes In Vitro and In Vivo

Example 15. Synthesis of GalNAc-Lipids 1042, 1043 and 1044

Compound 2002 was prepared according to reported procedure (OrganicLett., 2010, 12, 5262). Compound 2002 (1 mol eq), acrylonitrile (4.6 moleq) and 5M aq. NaOH (0.166 vol) and THF (10.0 vol) stirred at ambienttemperature for 48 h. The reaction mixture was concentrated and theresidue was dissolved in ethyl acetate (EtOAc, 5.0 vol) and washed withwater and brine. The organic layer was concentrated and purified bycolumn chromatography using EtOAc/MeOH eluent to obtain compound 2003 asa pale yellow liquid (yield: 50%).

Compound 2003 (1 mol eq.) and Raney-Ni (200% w/w) was suspended in 1:125% aq. Ammonia/water (10.0 vol) and hydrogenated at 50 kg/cm³ pressure.The reaction mixture was filtered through celite and concentrated toobtain compound 2004 as pale yellow liquid (yield: 84%).

The amine 2004 (1 mol eq.), compound 2005 (J. Am. Chem. Soc. 2014, 136,16958; 3.6 mol eq.) were stirred with EDC.HCl (4 mol eq.), HOBt (0.1 moleq) and DIEA (10 mol eq) in DMF (10 vol) at 0° C.-RT for 16 h. Thereaction mixture was slowly transferred to ice-water and top layer wasdecanted. Residue was dissolved in EtOAc, washed with 5% aq. citric acidfollowed 5% aq. Na₂CO₃ and brine. Organic layer was concentrated toobtain crude compound as a foamy solid. The crude thus obtained was thenpurified by column chromatography to obtain the desired compound 2006(52%).

Compound 2006 (1 mol eq.) was stirred with trifluoroacetic acid (4 vol.)in dichloromethane, 0° C.-RT for 24 h. The reaction mixture wasconcentrated to remove volatiles; residue was co-distilled with toluene(2 vol×2). Residue was dissolved in methanol (1 vol) and n-hexane (10vol); top layer was decanted and the residue was dissolved indichloromethane. Evaporated solvents and volatiles in vacuo to obtaincompound 2007 as a colorless paste (yield: 100%).

Compound 2008 (1 mol eq.) and 4-nitrophenyl chloroformate (4 mol eq.)were stirred in dichloromethane (10 vol) in the presence of pyridine (4mol eq.) at ambient temperature for 4 h. The reaction mixture wasevaporated in vacuo and the residue was purified column chromatographyto obtain compound 2009.

Compound 2009 (1 mol eq.) was stirred with the amine 2010 (1.5 mol eq.)in dichloromethane (10 vol) in the presence of pyridine (2 mol eq. atambient temperature overnight. The reaction mixture was diluted withwater. The product was extracted into dichloromethane and concentratedto dryness. The residue was purified by column chromatography to obtaincompound 2011 (yield: 81%). Treatment of 2011 (1 mol eq.) with formicacid (5 vol) in dichloromethane at ambient temperature for 6h. Solventand volatiles were removed in vacuo. The residue was washed with toluenetwice and with diethyl ether to obtain compound 2012 (yield: 80%).

Compound 2009 (1 mol eq.) was stirred with the amine 2013 (1.5 mol eq.)in dichloromethane (10 vol) in the presence of pyridine (2 mol eq.) atambient temperature overnight. The reaction mixture was diluted withwater. The product was extracted into dichloromethane and concentratedto dryness. The residue was purified by column chromatography to obtaincompound 2014 (yield: 65%). Treatment of 2014 (1 mol eq.) with formicacid (5 vol) in dichloromethane at ambient temperature for 6h. Solventand volatiles were removed in vacuo. The residue was washed with toluenetwice and with diethyl ether to obtain compound 2015 (yield: 87%).

Compound 2009 (1 mol eq.) was stirred with the amine 2016 (1.5 mol eq.)in dichloromethane (10 vol) in the presence of pyridine (2 mol eq.) atambient temperature overnight. The reaction mixture was diluted withwater. The product was extracted into dichloromethane and concentratedto dryness. The residue was purified by column chromatography to obtaincompound 2017 (yield: 51%). Treatment of 2017 (1 mol eq.) with formicacid (5 vol) in dichloromethane (2 vol) at ambient temperature for 6h.Solvent and volatiles were removed in vacuo. The residue was washed withtoluene twice and with diethyl ether to obtain compound 2018 (yield:87%).

Compound 2007 (1 mol eq.) and compound 2015 (1.1 mol eq) were mixed withHBTU (1.2 mol eq.), HOBt (0.1 mol eq.) and DIEA (3 mol eq) indichloromethane (10 vol) at 0° C.-RT under stirring for 2 h. Thereaction mixture was washed with water and the organic layer wasconcentrated in vacuo. The crude product was then purified by silica gelcolumn chromatography to obtain compound 2020 (yield: 35%). To astirring solution of compound 2020 (0.7 g, 0.19 mmol) in ethanol (3 mL)was added aqueous ammonia (6 mL) at room temperature and the resultingreaction mixture was warmed at 40° C. for 48 h. Reaction mixture isconcentrated under reduced pressure at 40° C. The residue obtained istriturated with diethyl ether (2×5 mL) and acetonitrile (3×5 mL), driedunder vacuum pressure to afford 1043 as off white solid (200 mg, yield:38.87%). ¹H NMR (400 MHz, DMSO-d₆): δ 7.70-7.80 (m, 2H), 7.60 (d, J=9.2Hz, 2H), 7.05-7.15 (m, 4H), 4.44-4.57 (m, 9H), 4.20 (d, J=8.4 Hz, 3H),3.86-3.98 (m, 2H), 3.62-3.68 (m, 9H), 3.46-3.55 (m, 55H), 3.25-3.39 (m,18H), 3.02-3.08 (m, 8H), 2.02 (t, J=6.8 Hz, 6H), 1.78 (s, 9H), 1.56-1.69(m, 12H), 1.40-1.48 (m, 17H), 1.22 (s, 56H), 0.82-0.85 (m, 6H); HRMS(ESI-TOF) m/z: [M+H]⁺ and [M+Na]⁺ calculated for 2598.71 and 2620.7;found 2598.72 and 2620.73.

Compound 2007 (1 mol eq.) and compound 2018 (1.1 mol eq) were mixed withHBTU (1.2 mol eq.), HOBt (0.1 mol eq.) and DIEA (3 mol eq) indichloromethane (10 vol) at 0° C.-RT under stirring for 2 h. Thereaction mixture was washed with water and the organic layer wasconcentrated in vacuo. The crude product was then purified by silica gelcolumn chromatography to obtain compound 2021 (yield: 30%). To a stirredsolution of compound 2021 (0.8 g, 0.17 mmol) in ethanol (3 mL) was addedaqueous ammonia (6 mL) at room temperature and the resulting reactionmixture was warmed at 40° C. for 48 h. Reaction mixture was concentratedunder reduced pressure at 40° C. The residue obtained was trituratedwith diethyl ether (2×5 mL) and acetonitrile (3×5 mL), dried undervacuum pressure to afford Compound 1044 as off white solid (450 mg,yield: 70%). ¹H NMR (400 MHz, DMSO-d₆): δ 7.71-7.74 (m, 1H), 7.61 (d,J=8.8 Hz, 1H), 7.10-7.14 (m, 1H), 4.52-4.62 (m, 2H), 4.46 (d, J=4.4 Hz,1H), 4.19 (d, J=8.4 Hz, 1H), 3.86-3.98 (m, 1H), 3.61-3.68 (m, 4H),3.46-3.55 (m, 57H), 3.27-3.42 (m, 8H), 3.0-3.15 (m, 3H), 2.0-2.03 (m,2H), 1.78-1.81 (m, 3H), 1.65-1.69 (m, 2H), 1.56-1.59 (m, 2H), 1.38-1.49(m, 6H), 1.15-1.25 (m, 23H), 0.81-0.85 (m, 2H); HRMS (ESI-TOF) m/z:[M+NH4]⁺ calculated for 3672.34; found 3672.37.

To a stirred solution of 2019 (850 mg, 00274 mmol) in ethanol (3 mL) wasadded aqueous ammonia (6 mL) at room temperature and the resultingreaction mixture was warmed at 40° C. for 48 h. Reaction mixture wasconcentrated under reduced pressure at 40° C. The residue obtained wastriturated with diethyl ether (5×15 mL) and acetonitrile (5×10 mL),dried under vacuum pressure to afford 1042 as off white solid (400 mg,yield: 67.5%). ¹H NMR (400 MHz, DMSO-d₆): δ 7.71 (t, J=5.6 Hz, 3H), 7.59(d, J=9.2 Hz, 3H), 7.12-7.15 (m, 2H), 4.52-4.57 (m, 6H), 4.44 (d, J=4.0Hz, 3H), 4.20 (d, J=8.4 Hz, 3H), 3.86-3.98 (m, 2H), 3.62-3.68 (m, 9H),3.45-3.53 (m, 22H), 3.31-3.39 (m, 19H), 3.02-3.10 (m, 9H), 2.02 (t,J=7.2 Hz, 6H), 1.78 (s, 9H), 1.64-1.70 (m, 6H), 1.54-1.61 (m, 6H),1.38-1.49 (m, 18H), 1.21 (s, 62H), 0.82-0.85 (m, 6H); HRMS (ESI-TOF)m/z: [M+H]⁺ calculated for 2158.44; found 2158.45.

Example 16. Synthesis of GalNAc-Lipids 1002, 1003 and 1004

Compound 2022 (1 mol eq.) and compound 2022A (1 mol eq.) were stirredwith EDC.HCl (1.1 mol eq.) in the presence of DIEA (2 mol eq.) and HOBt(0.1 mol eq.) in DMF at 0° C.-RT for 16 h. The reaction mixture wasslowly poured into ice-water and top layer was decanted. The residue wasdissolved in EtOAc and washed with 5% aq. citric acid, 5% aq. Na₂CO₃followed by water and brine wash. The organic layer was concentrated toobtain compound 2023 as a foamy solid (yield: 88.7%). The crude productthus obtained could be used for the next step without furtherpurification.

Compound 2023 (1 mol eq.) was stirred with trifluoroacetic acid (4 vol)in dichloromethane (4 vol) at 0° C.-RT for 24 h. The reaction mixturewas concentrated in vacuo to remove volatiles, and the residue wasco-distilled with toluene. The residue thus obtained was dissolved inmethanol (1 vol) and 10 vol of n-hexane was added. Top layer wasdecanted and the gummy mass was dissolved in dichloromethane andevaporated to get compound 2024 as a colorless paste (yield: 94.3%).

Compound 2024 (1 mol eq.) and compound 2005 (3.6 mol eq.) were stirredwith EDC.HCl (4 mol eq.) in the presence of DIEA (10 mol eq.) and HOBt(0.1 mol eq.) in DMF (10 vol) at 0° C.-RT for 16 h. The reaction mixturewas slowly poured into ice-water and top layer was decanted. The residuewas dissolved in EtOAc and washed with 5% aq. citric acid, 5% aq. Na₂CO₃followed by water and brine wash. The organic layer was concentrated toa foamy solid, which was then purified by column chromatography toobtain compound 2025 as a foamy solid (yield: 70%).

Compound 2025 (1 mol eq.) was suspended on 105 Pd—C in THF:IPA (1:3, 10vol) and hydrogenated at normal pressure. The reaction mixture wasfiltered through a celite bed. The filtrate was concentrated in vacuo toan off-white solid, which was subsequently purified by columnchromatography to afford compound 2026 (yield: 58%).

Compound 2026 (1 mol eq.) and compound 2012 (1.1 mol eq.) were stirredwith HBTU (1.2 mol eq.) in the presence of DIEA (3 mol eq.) and HOBt(0.1 mol eq.) in dichloromethane (10 vol) at 0° C.-RT for 2 h. Thereaction mixture was washed with water and the organic layer wasconcentrated to get crude compound as pale brown foamy solid. Columnchromatographic purification of the crude afforded compound 2027 (yield:60.7%). To a stirred solution of 2027 (1.4 g, 0.48 mmol) in methanol(14.0 mL) was added solution of sodium methoxide (26.0 mg, 0.48 mmol) inmethanol (1 mL) at 0° C.±5° C. and the resulting reaction mixture waswarmed to room temperature for 2 h. Reaction mixture was diluted withDCM (5.0 mL) and acidified with resin Dowex to pH ˜5 to 6. Reactionmixture was filtered through Buchner funnel and filtrate wasconcentrated under reduced pressure at 40° C. The residue obtained wastriturated with diethyl ether (5×20 mL) and acetonitrile (5×10 mL),dried under vacuum pressure to afford 1003 as off white solid (0.5 g,yield: 52.9%). ¹H NMR (400 MHz, DMSO-d₆): δ 8.11 (d, J=8 Hz, 1H), 7.83(t, J=5.2 Hz, 1H), 7.69-7.74 (m, 2H), 7.62 (d, J=8.8 Hz, 3H), 7.18 (t,J=5.6 Hz, 1H), 4.52-4.60 (m, 1H), 4.20-4.25 (m, 3H), 3.80-4.05 (m, 19H),3.64-3.74 (m, 10H), 3.42-3.52 (m, 17H), 3.25-3.40 (m, 16H), 2.97-3.10(m, 9H), 2.30-2.41 (m, 2H), 2.01-2.08 (m, 6H), 1.79 (s, 9H), 1.42-1.50(m, 21H), 1.22 (s, 61H), 0.82-0.86 (m, 6H); HRMS (ESI-TOF) m/z: [M+H]⁺and [M+Na]⁺ calculated for 1951.31 and 1973.31; found 1951.31 and1973.29.

Compound 2026 (1 mol eq.) and compound 2015 (1.1 mol eq.) were stirredwith HBTU (1.2 mol eq.) in the presence of DIEA (3 mol eq.) and HOBt(0.1 mol eq.) in dichloromethane (10 vol) at 0° C.-RT for 2 h. Thereaction mixture was washed with water and the organic layer wasconcentrated to get crude compound as pale brown foamy solid. Columnchromatographic purification of the crude afforded compound 2028 (yield:68.7%). To a stirred solution of 2028 (1.2 g, 0.36 mmol) in ethanol (3.0mL) was added aqueous ammonia (6.0 mL) at room temperature and theresulting reaction mixture was warmed at 40° C. for 48 h. Reactionmixture was concentrated under reduced pressure at 40° C. The residueobtained was triturated with diethyl ether (3×10 mL) and acetonitrile(4×10 mL), dried under vacuum pressure to afford compound 1002 as offwhite solid (470 mg, yield: 54.5%). ¹H NMR (400 MHz, DMSO-d₆): δ 8.09(d, J=8 Hz, 1H), 7.81 (t, J=5.2 Hz, 1H), 7.67-7.72 (m, 2H), 7.60 (d,J=9.2 Hz, 3H), 7.13 (t, J=5.6 Hz, 1H), 4.52-4.58 (m, 7H), 4.44 (d, J=4.0Hz, 3H), 4.20 (d, J=8.4 Hz, 3H), 3.86-3.98 (m, 3H), 3.57-3.71 (m, 10H),3.42-3.52 (m, 55H), 2.94-3.11 (m, 8H), 2.0-2.06 (m, 6H), 1.78 (s, 9H),1.35-1.75 (m, 21H), 1.22 (s, 60H), 0.80-0.85 (m, 6H); HRMS (ESI-TOF)m/z: [M+H]⁺ calculated for 2391.57; found 2391.58.

Compound 2026 (1 mol eq.) and compound 2018 (1.1 mol eq.) were stirredwith HBTU (1.2 mol eq.) in the presence of DIEA (3 mol eq.) and HOBt(0.1 mol eq.) in dichloromethane (10 vol) at 0° C.-RT for 2 h. Thereaction mixture was washed with water and the organic layer wasconcentrated to get crude compound as pale brown foamy solid. Columnchromatographic purification of the crude afforded compound 2029 (yield:68.7%). To a stirred solution of 2029 (0.55 g, 0.12 mmol) in ethanol(2.0 mL) was added aqueous ammonia (4.0 mL) at room temperature and theresulting reaction mixture was warmed at 40° C. for 48 h. Reactionmixture was concentrated under reduced pressure at 40° C. The residueobtained was triturated with diethyl ether (2×5 mL) and acetonitrile(3×5 mL), dried under vacuum pressure to afford 1004 as off white solid(230 mg, yield: 53.17%). ¹H NMR (400 MHz, DMSO-d₆): δ 8.11 (d, J=10 Hz,1H), 7.85-7.95 (m, 1H), 7.70-7.80 (m, 1H), 7.62 (d, J=11.6 Hz, 2H), 7.14(bs, 1H), 4.47-4.57 (m, 8H), 4.21 (d, J=10.8 Hz, 2H), 3.95-4.05 (m, 3H),3.86-3.96 (m, 3H), 3.63-3.67 (m, 13H), 3.28-3.38 (m, 30H), 2.95-3.15 (m,10H), 1.95-2.15 (m, 6H), 1.79 (s, 9H), 1.35-1.45 (m, 20H), 1.22 (s,52H), 0.85-0.95 (m, 6H); HRMS (ESI-TOF) m/z: [M+H]⁺ calculated for3448.20; found 3448.21.

Example 17. Synthesis of GalNAc-Lipids 1013 and 1052

Cholesteryl chloroformate was reacted with compound 2013 in the presenceof base in dichloromethane afforded compound 2030. Compound 2030 wastreated with formic acid in THF to afford compound 2031. Compound 2031was reacted with compound 2007 as described in Example 16 to yieldcompound 2032. To a stirred solution of 2032 (880 mg, 0.264 mmol) inethanol (3 mL) was added aqueous ammonia (6 mL) at room temperature andthe resulting reaction mixture was warmed at 40° C. for 48 h. Reactionmixture was concentrated under reduced pressure at 40° C. The residueobtained was triturated with diethyl ether (10×10 mL), dried undervacuum pressure to afford 1052 as off white solid (500 mg, yield:79.5%). ¹H NMR (400 MHz, DMSO-d₆): δ 7.65-7.75 (m, 1H), 7.81 (bs, 1H),7.70-7.80 (m, 1H), 7.59-7.61 (d, J=8.4 Hz, 2H), 7.0-7.11 (bs, 3H), 5.30(s, 1H), 4.50-4.70 (m, 6H), 4.40-4.50 (m, 3H), 4.26-4.44 (m, 3H),3.61-3.67 (m, 8H), 3.35-3.47 (m, 45H), 2.95-3.07 (m, 8H), 1.95-2.01 (m,6H), 1.76 (s, 13H), 1.30-1.47 (m, 26H), 1.05-1.15 (m, 8H), 0.95-1.0 (m,4H), 0.8-0.95 (m, 8H), 0.65 (s, 3H); HRMS (ESI-TOF) m/z: [M+H]⁺calculated for 2374.91; found 2374.37.

To a stirred solution of 2033 (730 mg, 0.23 mmol) in ethanol (3 mL) wasadded aqueous ammonia (6 mL) at room temperature and the resultingreaction mixture was warmed at 40° C. for 48 h. Reaction mixture wasconcentrated under reduced pressure at 40° C. The residue obtained wastriturated with diethyl ether (5×10 mL), dried under vacuum pressure toafford 1013 as off white solid (500 mg, yield: 97.9%). ¹H NMR (400 MHz,DMSO-d₆): δ 8.08 (d, J=7.2 Hz, 1H), 7.80-7.90 (m, 1H), 7.65-7.75 (m,2H), 7.60 (d, J=8.4 Hz, 2H), 7.0-7.11 (m, 3H), 5.30 (s, 1H), 4.51-4.55(m, 6H), 4.44-4.45 (m, 2H), 4.17-4.27 (m, 3H), 3.62-3.70 (m, 8H),3.37-3.60 (m, 45H), 2.95-3.15 (m, 8H), 1.95-2.10 (m, 6H), 1.76 (s, 9H),1.25-1.75 (m, 30H), 1.05-1.20 (m, 9H), 0.80-1.0 (m, 12H), 0.45-0.55 (m,3H); HRMS (ESI-TOF) m/z: [M+H]⁺ calculated for 2181.67; found 2182.32.

Example 18. Synthesis of GalNAc-Lipids 1014 and 1053

To a stirred solution of 2037 (0.9 g, 0.21 mmol) in ethanol (3 mL) wasadded aqueous ammonia (6 mL) at room temperature and the resultingreaction mixture was warmed at 40° C. for 48 h. Reaction mixture wasconcentrated under reduced pressure at 40° C. The residue obtained wastriturated with diethyl ether (6×10 mL), dried under vacuum pressure toafford 1014 (300 mg, yield: 42.97%) as off white solid. ¹H NMR (400 MHz,DMSO-d₆): δ 8.10 (d, J=8.0 Hz, 1H), 7.83 (t, J=6.8 Hz, 1H), 7.72 (q,J=5.6 Hz, 2H), 7.62 (d, J=8.4 Hz, 3H), 7.02 (t, J=5.2 Hz, 1H), 5.33 (bs,1H), 4.53-4.59 (m, 7H), 4.46 (d, J=4.0 Hz, 3H), 4.20-4.32 (m, 4H),3.63-3.72 (m, 11H), 3.30-3.60 (m, 157H), 2.95-3.15 (m, 9H), 2.26-2.38(m, 3H), 2.03-2.08 (m, 6H), 1.90-2.0 (m, 2H), 1.75-1.86 (m, 13H),1.30-1.60 (m, 31H), 0.95-1.15 (m, 13H), 0.83-0.90 (m, 10H), 0.64 (s,3H); HRMS (ESI-TOF) m/z: [M+H]⁺ calculated for 3238.94; found 3238.95.

To a stirred solution of 2036 (1.2 mg, 0.264 mmol) in ethanol (6 mL) wasadded aqueous ammonia (12 mL) at room temperature and the resultingreaction mixture was warmed at 40° C. for 48 h. Reaction mixture wasconcentrated under reduced pressure at 40° C. The residue obtained wastriturated with diethyl ether (12×10 mL), dried under vacuum pressure toafford 1053 as off white wax (500 mg, yield: 53.0%). ¹H NMR (400 MHz,DMSO-d₆): δ 7.70-7.75 (m, 2H), 7.59 (d, J=8.8 Hz, 2H), 6.95-7.15 (m,4H), 5.32 (bs, 1H), 4.51-4.56 (m, 2H), 4.44-4.45 (m, 1H), 4.18 (d, J=8.4Hz, 1H), 3.61-3.67 (m, 4H), 3.34-3.67 (m, 51H), 3.29-3.34 (m, 6H),3.03-3.05 (m, 3H), 2.0-2.04 (m, 3H), 1.88-1.96 (m, 2H), 1.76 (s, 4H),1.65-1.75 (m, 2H), 1.55-1.65 (m, 3H), 1.30-1.50 (m, 8H), 1.0-1.20 (m,3H), 0.90-0.93 (m, 1H), 0.86 (d, J=6.4 Hz, 1H), 0.81 (dd, J=6.4 Hz,J=1.6 Hz, 2H), 0.6 (s, 1H); HRMS (ESI-TOF) m/z: [M+H]⁺ calculated for3446.05; found 3446.08.

Example 19. Synthesis of GalNAc-Lipids 1062 and 1065

Compound 2038 is reacted with 4-nitrophenyl chloroformate in thepresence of a base to form the corresponding 4-nitrophenyl carbonate.The carbonate thus formed is reacted with compound 2016 to yieldcompound 2039. Compound 2039 is treated with formic acid to obtaincompound 2040. Compound 2040 is coupled to 2007 under peptide couplingconditions as described in Example 15 afforded compound 2041. Compound2041 is treated with NaOMe followed by work-up and purification asdescribed in Example 15/16 afforded compound 1062.

Compound 1065 is prepared from compounds 2040 and 2026 as describedabove.

Example 20. Synthesis of GalNAc-Lipids 1062 and 1065

Compound 2038 is reacted with 4-nitrophenyl chloroformate in thepresence of a base to form the corresponding 4-nitrophenyl carbonate.The carbonate thus formed is reacted with compound 2013 in the presenceof base in dichloromethane afforded compound 2043. Compound 2043 istreated with formic acid in THF to afford compound 2044. Compound 2044is reacted with compound 2007 as described in Example 16 to yieldcompound 2045. Compound 2045 is treated with NaOMe and followed similarwork-up and purification afforded compound 1062.

Compound 1065 is prepared from compounds 2044 and 2026 as describedabove. Compounds 1083, 1084 and 1085 may also be prepared from galactosein a similar manner in accordance with preparation of 1004 as describedabove.

Example 20A

Exemplary synthesis of GallNAc-Lipid 1076: Jo a suspension of 3000 (4.6g, 9.5 mmol) in DCM (46 mL) was added pyridine (3.0 mL) drop wise atroom temperature (RT) over a period of 10 min. To the above solution,p-Nitrophenylchloroformate (7.66 g, 38.0 mmol) was added portion wiseand the resulting suspension stirred at RT for 1 h. Reaction mixture wasconcentrated under reduced pressure and the crude mass was purified bysilica gel column chromatography (CombiFlash rf) using 8% EtOAc inHexane as eluent to yield 3001 as color less liquid (3.01 g, yield:48.78%). ¹H NMR (400 MHz, CDCl₃): δ 8.26-8.29 (m, 2H), 7.37-7.41 (m,2H), 5.44-5.48 (m, 1H), 4.31-4.35 (m, 1H), 3.71-3.73 (m, 1H), 3.44-3.61(m, 6H), 1.53-1.60 (m, 4H), 1.25-1.31 (m, 8H), 0.86-0.89 (m, 6H). To astirred solution of 3001 (3 g, 4.6 mmol) in DCM (30 mL) was addedpyridine (0.74 mL) and 2016 (11.98 g, 6.9 mol) at RT. The reaction wascontinued for 12 h at RT and the reaction mixture was concentrated underreduced and the crude compound was purified by neutral alumina columnchromatography (CombiFlash rf) using 80% EtOAc in Hexane as eluent toafford 3002 as off white solid (5.35 g, yield: 51.89%). ¹H NMR (400 MHz,DMSO-d₆): δ 7.13 (t, J=5.6 Hz, 1H), 3.86-3.99 (m, 2H), 3.47-3.57 (m,4H), 3.30-3.49 (m, 146H), 3.07-3.11 (m, 2H), 2.39-2.49 (m, 2H),1.45-1.55 (m, 4H), 1.45 (s, 9H), 1.20-1.45 (m, 43H), 0.75-0.85 (m, 6H).

Formic acid (35.0 mL) was added to a stirred solution of 3002 (5.0 g,2.2 mmol) in DCM (10 mL) at ice cold temperature and the resultingreaction was stirred at RT for 6 h. The reaction mixture wasconcentrated to remove formic acid and co-distilled with toluene underreduced pressure to get the desired 3003 as off-white solid (4.28 g,yield: 89.1%). 3003 was used as is in the next step. ¹H NMR (400 MHz,CDCl₃): δ 5.27 (bs, 1H), 4.06-4.20 (m, 4H), 3.75-3.85 (m, 3H), 3.50-3.70(m, 144H), 3.30-3.50 (m, 6H), 2.59 (t, J=6 Hz, 2H), 1.53-1.56 (m, 4H),1.20-1.30 (m, 45H), 0.80-0.90 (m, 6H). To a stirred solution of 3003(3.32 g, 1.5 mmol) in DCM (32.0 mL) was added HOBt (0.02 g, 0.15 mmol)and HBTU (0.71 g, 1.8 mmol) at ice cold temperature followed by theaddition of DIPEA (0.78 mL, 4.5 mmol). A solution of 2026 (3.2 g, 1.5mmol) in DCM (15.0 mL) was added to above reaction at RT and theresulting reaction mixture was stirred for 1 h at RT. Water (30.0 mL)was added to the reaction mixture and extracted with DCM (2×30.0 mL).The combined organic layers washed with saturated aqueous sodiumbicarbonate solution (50.0 mL) followed by brine (50.0 mL), dried overanhydrous sodium sulfate. The organic layer was filtered and filtratewas evaporated under reduced pressure. The crude was purified by silicagel column chromatography (CombiFlash rf) using 10% MeOH in DCM aseluent to afford 3004 (2.9 g, yield: 45.24%) as grey semisolid. ¹H NMR(400 MHz, DMSO-d₆): δ 7.97-8.13 (m, 4H), 7.88-7.92 (m, 12H), 7.83 (t,J=4.8 Hz, 1H), 7.66-7.73 (m, 11H), 7.62 (t, J=7.2 Hz, 3H), 7.53-7.58 (m,9H), 7.47 (t, J=7.6 Hz, 6H), 7.37 (t, J=8 Hz, 6H), 7.15 (t, J=6 Hz, 1H),5.73-5.75 (m, 3H), 5.35 (dd, J=10.8 Hz, J=2.8 Hz, 3H), 4.71 (d, J=8.4Hz, 3H), 4.45-4.57 (m, 1H), 4.41-4.45 (m, 6H), 4.22-4.35 (m, 6H),3.30-4.0 (m, 12H), 3.08-3.20 (m, 9H), 2.03-2.07 (m, 1.68 (s, 11H),1.42-1.49 (m, 22H), 1.21-1.30 (m, 48H), 0.80-0.90 (m, 6H).

Aqueous ammonia (16.8 mL) was added to a stirred solution of 3004 (2.8g, 0.69 mmol) in ethanol (8.4 mL) at RT and the resulting reactionmixture was stirred at 40° C. for 48 h. Reaction mixture wasconcentrated under reduced pressure. The resultant residue wastriturated with diethyl ether (10×50 mL) and the residue was dried undervacuum pressure to afford 1076 as pale yellow solid (1.85 g, yield:80.43%). ¹H NMR (400 MHz, DMSO-d₆): δ 8.11 (d, J=5.6 Hz, 1H), 7.81-7.84(m, 1H), 7.69-7.74 (m, 2H), 7.61 (d, J=8.8 Hz, 3H), 7.14 (t, J=5.6 Hz,1H), 4.54-4.59 (m, 7H), 4.46-4.47 (m, 3H), 4.20 (d, J=8.4 Hz, 3H),3.86-3.99 (m, 2H), 3.62-3.68 (m, 10H), 3.30-3.50 (m, 159H), 2.96-3.11(m, 11H), 2.0-2.07 (m, 7H), 1.78 (s, 9H), 1.41-1.47 (m, 26H), 1.18-1.22(m, 54H), 0.80-0.85 (m, 8H); HRMS (ESI-TOF) m/z: [M+H]⁺ calculated for3336.08; found 3336.0.

Example 20B: Exemplary Synthesis of 1079 (Scheme 19)

To a stirred solution of stearic acid (0.43 g, 1.51 mmol) in DCM (8.6mL) was added HOBt (0.02 g, 0.15 mmol) at RT. To this reaction mixturewas added HBTU (0.72 g, 1.81 mmol) at ice cold temperature followed byaddition of DIPEA (0.78 mL, 4.53 mmol). A solution of 2016 (2.61 g, 1.51mmol) in DCM (2.1 mL, 5 vol) was added at ice cold temperature to theabove reaction mixture and the resulting mixture was stirred for 4 h atRT. Water (30.0 mL) was added to reaction and extracted with DCM (2×30mL). The combined organic layers was washed with brine (30.0 mL), driedover anhydrous sodium sulfate, and filtered. The filtrate was evaporatedunder reduced pressure and the crude was purified by silica gel columnchromatography (CombiFlash rf) using 10% MeOH in DCM as eluent to afford3005 as off white solid (2.31 g, Yield: 76.74%). ¹H NMR (400 MHz,CDCl₃): δ 6.30-6.40 (bs, 1H), 3.38-3.72 (m, 151H), 2.68 (bs, 3H), 2.49(t, J=8.8 Hz, 2H), 2.19 (t, J=9.6 Hz, 2H), 1.61-1.64 (m, 4H), 1.45 (s,9H), 1.20-1.45 (m, 29H), 0.80-0.85 (m, 3H).

Formic acid (14.7 mL) was added to a stirred solution of 3005 (2.1 g,1.0 mmol) in DCM (10.5 mL) at ice cold temperature. The resultingreaction mixture was stirred at room temperature for 12 h. The reactionmixture was concentrated to remove formic acid and co-distilled withtoluene (3×) under reduced pressure. The residue was triturated withdiethyl ether (21.0 mL), filtered and resultant residue was dried undervacuum pressure to obtain 3006 as off-white solid (1.81 g, yield:88.72%). ¹H NMR (400 MHz, CDCl₃): δ 6.25 (bs, 1H), 3.54-3.81 (m, 144H),3.42-3.47 (m, 3H), 2.59 (t, J=6.4 Hz, 2H), 2.17 (t, J=7.6 Hz, 2H),1.40-1.63 (m, 4H), 1.20-1.45 (m, 27H), 0.80-0.85 (m, 3H).

To a stirred solution of 3006 (0.58 g, 0.29 mmol) in DCM (8.0 mL) wasadded HOBt (0.038 g, 0.028 mmol) at RT. To this reaction mixture wasadded HBTU (0.13 g, 0.34 mmol) at ice cold temperature followed by DIPEA(0.14 mL, 0.85 mmol). A solution of 2026 (0.6 g, 0.28 mmol) in DCM (3.0mL) was added at ice cold temperature to above and the resultingreaction mixture was stirred for 2 h at RT. To the reaction mixture wasadded water (25 mL) and extracted with DCM (2×20 mL). The combinedorganic layer was washed with brine (30 mL), dried over anhydrous sodiumsulphate, and filtered. The filtrate was evaporated under reducedpressure and crude was purified by silica gel column chromatography(CombiFlash rf) using 10% MeOH in DCM as eluent to afford 3007 as greysemisolid (0.76 g, yield: 66.66%). ¹H NMR (400 MHz, DMSO-d₆): δ 8.74(bs, 1H), 8.11 (d, J=8 Hz, 1H), 7.99 (d, J=9.2 Hz, 3H), 7.88-7.92 (m,13H), 7.78-7.85 (m, 2H), 7.66-7.74 (m, 11H), 7.62 (t, J=7.2 Hz, 3H),7.52-7.58 (m, 9H), 7.47 (t, J=8 Hz, 6H), 7.39 (t, J=8 Hz, 6H), 5.73-5.75(m, 3H), 5.35 (dd, J=14 Hz, J=3.2 Hz, 3H), 4.72 (d, J=8.4 Hz, 3H),4.50-4.57 (m, 1H), 4.41-4.45 (m, 6H), 4.24-4.33 (m, 6H), 3.76-3.79 (m,3H), 3.43-3.61 (m, 175H), 3.32-3.38 (m, 3H), 3.08-3.16 (m, 8H), 2.90-3.0(m, 5H), 2.0-2.07 (m, 8H), 1.68 (s, 12H), 1.40-1.49 (m, 21H), 1.21-1.27(m, 62H), 0.80-0.90 (m, 3H).

To a stirred solution of 3007 (0.75 g, 0.69 mmol) in ethanol (2.2 mL)was added aqueous ammonia (4.5 mL) at RT and the resulting reaction wascontinued at 40° C. for 48 h. Reaction mixture was concentrated underreduced pressure and the crude was triturated with diethyl ether (5×20mL). The solid residue after filtration was dried under reduced pressureto afford 1079 as off white solid (0.35 g, yield: 60.8%). ¹H NMR (400MHz, DMSO-d₆): δ 8.11 (d, J=7.6 Hz, 1H), 7.80-7.85 (m, 2H), 7.70-7.75(m, 2H), 7.63 (d, J=8.4 Hz, 3H)), 4.47-4.59 (m, 9H), 4.20 (d, J=8 Hz,3H), 3.62-3.68 (m, 12H), 3.49 (s, 141H), 2.97-3.16 (m, 11H), 2.0-2.03(m, 8H), 1.78 (s, 9H), 1.42-1.47 (m, 19H), 1.21 (s, 29H), 0.80-0.855 (m,3H); HRMS (ESI-TOF) m/z: [M+H]⁺ calculated for 3091.87; found 3092.75.

Example 20C: Exemplary Synthesis of 1078 (Scheme 20)

To a stirred solution of arachidic acid (0.46 g, 1.40 mmol) in DCM (10.0mL)) was added HOBt (0.018 g, 0.14 mmol) at RT. To this reaction mixturewas added HBTU (0.67 g, 1.68 mmol) at ice cold temperature followed byDIPEA (0.76 mL, 4.20 mmol). A solution of 2016 (2.54 g, 1.40 mmol) inDCM (5 mL) was added at ice cold temperature and the resulting reactionmixture was stirred for 4 h at RT. To the reaction mixture was addedwater (30.0 mL) and extracted with DCM (2×30 mL). The combined organiclayer was washed with brine (30.0 mL), dried over anhydrous sodiumsulphate and filtered. Filtrate was evaporated under reduced pressureand obtained crude was purified by silica gel column chromatographyusing 10% MeOH in DCM as eluent to afford 3008 as off white solid (2.52g, yield: 89.04%). ¹H NMR (400 MHz, CDCl₃): δ 6.38 (bs, 1H), 3.38-3.72(m, 132H), 2.47-2.57 (m, 4H), 2.19 (t, J=9.6 Hz, 1H), 1.61-1.64 (m, 1H),1.43 (s, 9H), 1.20-1.24 (m, 29H), 0.80-0.85 (m, 3H).

Formic acid (16.1 mL) was added to a stirred solution of 3008 (2.3 g,1.10 mmol) at ice cold temperature in DCM (11.5 mL) and the resultingreaction mixture was stirred for 12 h at room temperature. The reactionmixture was concentrated to remove formic acid and co-distilled withtoluene (3×) under reduced pressure and obtained residue was trituratedwith diethyl ether (23 mL), filtered and the residue was dried underhigh vacuum to yield 3009 as off-white solid (2.11 g, yield: 94.6%). ¹HNMR (400 MHz, CDCl₃): δ 6.29 (bs, 1H), 5.03 (bs, 3H), 3.54-3.82 (m,146H), 3.44-3.46 (m, 3H), 2.59 (t, J=6 Hz, 2H), 2.18 (t, J=7.6 Hz, 2H),1.59-1.63 (m, 2H), 1.25-1.35 (s, 32H), 0.80-0.85 (m, 3H).

To a stirred solution of 3009 (0.59 g, 0.29 mmol) in DCM (6 mL) wasadded HOBt (0.038 g, 0.028 mmol) at room temperature. To this reactionmixture was added HBTU (0.135 g, 0.34 mmol) at ice cold temperaturefollowed by DIPEA (0.14 mL, 0.85 mmol). A solution of 2026 (0.6 g, 0.28mmol) in DCM (3 mL) was added and the resulting reaction mixture wasstirred for 2 h at RT. To the reaction mixture was added water (25 mL)and extracted with DCM (2×20 mL). The combined organic layers werewashed with brine (30.0 mL), dried over anhydrous sodium sulphate, andfiltered. Filtrate was evaporated under reduced pressure and crude waspurified by silica gel column chromatography (CombiFlash rf) using 10%MeOH in DCM as eluent to afford 3010 as grey semisolid (0.91 g, yield:79.13%). ¹H NMR (400 MHz, DMSO-d₆): δ 8.11 (d, J=7.6 Hz, 1H), 7.97 (d,J=9.2 Hz, 3H), 7.90 (t, J=7.2 Hz, 12H), 7.78-7.82 (m, 2H), 7.66-7.70 (m,11H), 7.62-7.64 (m, 3H), 7.53-7.60 (m, 9H), 7.47 (t, J=8 Hz, 6H), 7.37(t, J=7.6 Hz, 6H), 5.73-5.74 (m, 4H), 5.35 (dd, J=11.2 Hz, J=2.8 Hz,3H), 4.72 (d, J=8.4 Hz, 3H), 4.50-4.57 (m, 1H), 4.41-4.47 (m, 6H),4.24-4.36 (m, 6H), 3.76-3.79 (m, 3H), 3.43-3.49 (m, 151H), 3.35-3.38 (m,3H), 2.96-3.18 (m, 10H), 2.30-2.35 (m, 1H), 2.0-2.06 (m, 8H), 1.68 (s,12H), 1.44-1.49 (m, 20H), 1.21-1.26 (m, 44H), 0.81-0.85 (m, 3H).

To a stirred solution of 3010 (0.87 g, 0.21 mmol) in ethanol (2.6 mL)was added aqueous ammonia (5.2 mL) at room temperature and the resultingreaction mixture was continued at 40° C. for 48 h. Purification wasperformed as described in 1079 synthesis to afford 1078 as off whitesolid (0.45 g, yield: 67.25%). ¹H NMR (400 MHz, DMSO-d₆): δ 8.10-8.12(m, 1H), 7.80-7.85 (m, 2H), 7.70-7.75 (m, 2H), 7.63 (d, J=7.6 Hz, 3H)),4.50-4.60 (m, 9H), 4.20 (d, J=8 Hz, 3H), 3.63-3.68 (m, 12H), 3.49 (s,162H), 2.97-3.16 (m, 17H), 2.0-2.03 (m, 9H), 1.78 (s, 9H), 1.42-1.47 (m,24H), 1.21 (s, 36H), 0.80-0.85 (m, 3H); HRMS (ESI-TOF) m/z: [M+H]⁺calculated for 3119.91; found 3120.8.

Example 21. Synthesis of mPEG2000-Cholesterol 504

mPEG-2000-NH₂ (1 mol eq.) was stirred with cholesteryl chloroformate(1001, 1 mol eq.) in the presence of pyridine (3 mol eq.) indichloromethane at ambient temperature for 18 h. The reaction mixturewas washed with water; solvents and volatiles were evaporated in vacuo.The crude mixture was subjected to silica gel column chromatographicpurification to obtain the desired compound 504 (yield: 22%). ELSD-HPLC99%; average mol. Wt. 2329; found: 2330.56. ¹H NMR (400 MHz, DMSO, d₆):δ ppm 7.01-6.98 (t, 1H), 5.35 (bs, 1H), 4.41-4.21 (m, 1H), 3.85-3.64 (m,1H), 3.68-3.35 (m, 194H), 3.23 (s, 3H), 3.18-2.95 (m, 2H), 2.42-2.15 (m,2H), 2.00-1.96 (m, 2H), 1.95-1.76 (m, 3H), 1.75-0.32 (m, 36H), 0.23-0.15(s, 3H).

Example 22. Guide RNA (gRNA) and mRNA for LNP Evaluation

The guide RNAs (gRNA) shown in Table 5 were synthesized under solidphase oligonucleotide synthesis and deprotection conditions usingcontrolled pore glass support and commercially available phosphoramiditemonomers and oligonucleotide synthesis reagents (Methods in MolecularBiology, 1993, 20, 81-114; ACS Chem. Biol. 2015, 10, 1181-1187,incorporated herein by reference in its entirety). The deprotected guideRNAs were purified by HPLC and the integrity of each guide RNA wasconfirmed by mass spectrometric analysis. The observed mass of eachguide RNA was conformed to calculated mass.

TABLE 5Single guide RNA (gRNA) used in the studies described in Examples 2-25gRNA Protospacer Protospacer SEQ ID Target* gRNA SEQ ID No (5′-3′)gRNA sequence (5′-3′)# NO PCSK9 GA055 104 CAGGTTCCATcsasgsGUUCCAUGGGAUGCUCUgUUUUAGagcu 121 GGGATGCTCTagaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAAcuugaaaaagugGcaccgagucggugcusususu PCSK9 GA010 105 GGCTGATGAgsgscsUGAUGAGGCCGCACAUGGUUUUAGAgcu 122 GGCCGCACATagaaauagcAAGUUAAAAUAAGGCUAGUCCGUU G AUCAacuugaaaaaguggcaccgagucggugcusususu PCSK9 GA255 106 CCCATACC cscscsAUACCUUGGAGCAACGGgUUUUA 123 TTGGAGCAGagcuaGaaauagcaaGUUaAaAuAaggCUaGUC ACGGcGUUAucAAcuuGaaaaaguGgcaccgAgUCggu gcusususu PCSK9 GA256 107 CCCATACCcscscsAUACCUUGGAGCAACGGgUUUUA 123 TTGGAGCAGagcuagaaauagcaaGUUaAaAuAaggcuaGUcc ACGGGUUAucAAcuugaaaaagugGcaccgagucggugcus ususu PCSK9 GA257 108 CCCATACCcscscsAUACCUUGGAGCAACGGgUUUUA 123 TTGGAGCAGagcuaGaaauagcaaGUUaAaAuAaggcuaGUcc ACGGGUUAucAAcuuGaaaaagugGcaccgagucggugcu sususu PCSK9 GA292 109 CCCATACCcscscsAUACUUGGAGCAACGGGUUUUAG 124 TTGGAGCA AGCUAGAAAUAGCAAGUUAAAAUAAGACGG GCUAGUCCGUUAUCAACUUGAAAAAG UGGCACCGAGUCGGUGCUsususu PCSK9 GA097 110CCCGCACC cscscsGCACCUUGGCGCAGCGGgUUUUA 125 TTGGCGCAGagcuagaaauagcaaGUUaAaAuAaggcuaGUcc GCGGGUUAucAAcuugaaaaagugGcaccgagucggugcus ususu ANGPTL3 GA258 111 GAGATACCgsasgsAUACCUGAGUAACUUUCgUUUUA 126 TGAGTAACGagcuaGaaauagcaaGUUaAaAuAaggCUaGUC TTTCcGUUAucAAcuuGaaaaaguGgcaccgAgUCggu gcusususu ANGPTL3 GA259 112 GAGATACCgsasgsAUACCUGAGUAACUUUCgUUUUA 126 TGAGTAACGagcuagaaauagcaaGUUaAaAuAaggcuaGUcc TTTCGUUAucAAcuugaaaaagugGcaccgagucggugcus ususu ANGPTL3 GA260 113 GAGATACCgsasgsAUACCUGAGUAACUUUCgUUUUA 126 TGAGTAACGagcuaGaaauagcaaGUUaAaAuAaggcuaGUcc TTTCGUUAucAAcuuGaaaaagugGcaccgagucggugcu sususu *The gRNAs were designed totarget mouse, rat, monkey and human PCSK9 and ANGPTL3 genes. #uppercaseand lowercase letters in the guide RNA sequence indicate nucleotidescarrying 2′-ribo (2′-OH) and 2′-O-methyl (2′-OMe) ribosugar moiety,respectively, and the letter ‘s’ indicates phosphorothioate (PS)linkage.mRNA encoding SpCas9, CBE, and ABE proteins

mRNA for SpCas9, CBE, and ABE were produced by different methods wellknown in the art. One of such methods used herein was in vitrotranscription (IVT) using T7 polymerase or additional RNA polymerasevariants. Typically, IVT of mRNA uses a linearized DNA template thatcomprises a T7 polymerase promoter, mRNA coding sequence (CDS), 3′ and5′ untranslated regions (UTRs), poly A tail, and additional replicationand transcription regulatory elements. Prior to IVT, the DNA templatewas in the form of a plasmid, PCR product, or additional double-strandedDNA construct. A typical IVT reaction includes T7 polymerase, DNAtemplate, RNase inhibitor, cap analog, inorganic pyrophosphatase, andnaturally occurring ribonucleotides (rNTPs) such as GTP, ATP, CTP, UTP,or substitutions of natural rNTPs with modified rNTPs such aspseudouridine, N1-methylpseudouridine, 5-methyl cytidine,5-methoxyuridine, N6-methyl adenosine, and N4-acetylcytidine. The capanalog was a dinucleotide or trinucleotide cap structure with the firstinitiating nucleotide containing standard 2′-hydroxyl group, 2′-O-methylgroup, or additional 2′ chemical modification. Cap analog also was addedafter the IVT reaction using a vaccinia capping enzyme. After IVT, insome cases DNase is added to the transcription mixture to remove DNAtemplate; alternatively, residual DNA was removed by ion exchange columnchromatography. Purification and concentration of mRNA were performedwith ion exchange chromatography, affinity chromatography,precipitation, ion-pairing reverse-phase chromatography, enzymaticreactions, size exclusion chromatography, and/or tangential flowfiltration. Similar IVT and purification process were used to producemRNA encoding SpCas9, CBE, and ABE; in all cases the DNA template,reaction conditions, and purification parameters were optimized for thespecific gene of interest. In some examples, capped and polyadenylatedmRNA were obtained from commercially sources (TriLink, for e.g.).

Example 23. Preparation of Lipid Nanoparticles (LNPs)

The LNPs used as reference in these studies are prepared according topublished procedures and are constituted from published LNP excipientsand genome editor mRNAs (Miller et al., Angew. Chem. Int. Ed. 2017, 56,1059-1063; Yin et al., Nature Biotechnology 2016, 34, 328-333) and guideRNAs (Chadwick et al., Arterioscler. Thromb. Vasc. Biol. 2017, 37,1741-1747; Rossidis et al., Nat. Med. 2018, 24, 1513-1518.doi:10.1038/s41591-018-0184-6; Ding et al., Circ Res. 2014, 115,488-492). The gRNA payload is selected from Table 5 and mRNA payloadsused for constituting these LNPs are prepared as described in Example 22or may be purchased commercially from third parties, for example, suchas TriLink BioTechnologies. The reference LNP A1 (Table 6) isconstituted from the published lipid 501 (Table 6), cholesterol, DSPCand PEG-lipid from Table 7 as described in the literature (Angew. Chem.Int. Ed. 2012, 51, 8529-8533). Similarly, the benchmark LNPs B1 and C1are constituted from lipids 502 (WO 2015/095340 A1) and 503 (MolecularTherapy 2018, 26, 1509-1519) respectively in combination withcholesterol, DSPC and PEG-DMG (506 and 507, Table 7) as summarized inTable 6. The reported genome editor nuclease mRNA and guide RNAs areused as payload for constituting the reference LNPs. In one approach theLNP formulations A1, B1 and C1 are made by co-formulating mRNA and guideRNA. In this co-formulation method mRNA to guide RNA ratio is variedfrom 10:1 to 1:10, that result in a series of LNPs for in vitro and invivo gene editing evaluation. In the second approach: guide RNA and mRNAare formulated separately using same lipid ratios as in Table 6 and thenpre-formulated LNPs with guide and mRNA are mixed together at variousratios to obtain a new series of LNPs for gene editing evaluation.

TABLE 6 Exemplary lipid compositions of LNPs A1, B1 and C1 Excipients, %mol LNP PEG- Formulation Lipid Lipid Cholesterol DSPC DMG A1 501 50 38.510  1.5 B1 502 45 44   9 2   C1 503 50 38.5 9 1.5

TABLE 7 Lipids and PEG-Lipids excipients used for LNP preparationsCompound number Structure 501

502

503

504

505

506

507

508

Example 24. Preparation of Hepatocyte Targeting LNPs Example 24-A

LNPs from Example 23 were reconstituted successively with GalNAc-lipid1002 and 1004 (Table 4) to obtain the desired hepatocyte targetedGalNAc-LNPs. A series of GalNAc-LNPs were generated by successivelyco-formulating 1002 or 1004 using the composition described in Table 6at different mol % (0.01 to 5 mol %) of each targeting lipids. Anotherset of LNPs were yielded when 1002 and 1004 was successively added atvarious mol % (0.01 to 5 mol %) to preformulated LNPs from Table 6 forin vitro and in vivo gene editing. Hepatocyte targeting GalNAc-Lipid(1002 or 1004) was added during the formulation as described in Tables8-13.

All LNPs prepared were stored either at 2-8° C. or −80° C. (Tables 6,8-13). Following formulation, LNPs were buffer exchanged andconcentrated. LNPs were buffer exchanged into buffers of varying ionicstrengths from 0 to 200 mM. In some cases, the buffer exchange wascarried out by PD-10 column, in others it was dialysis, and in otherinstances Tangential Flow Filtration (TFF) was used. In some cases, TFFwas used to concentrate the LNPs, and in other instances an amiconcentrifugation concentration column was used. LNPs were exchanged intoand concentrated in the final formulation buffer at pH 7 or 8.Cryoprotectant was added such that the final concentration ofcryoprotectant in the final formulation buffer is 0-500 mM. LNPs werewithout cryoprotectant were then stored at 2-8° C., and with cryoprotectwere frozen first and stored at −80° C.

Example 24-B

The targeting lipids of Example 24-A is then successively replaced withtargeting lipids 1042, 1043 and 1044 (Table 4), where the presentationof the ligand is different than those of 1002, 1003 and 1004. The lipidchain and tethers separating the ligand moieties are kept the same inboth series of targeting lipids. The same number of formulations withdifferent ratio of payloads (guide RNA and mRNA) and individualformulation of guide RNA and mRNA are also prepared as described inExample 23 for evaluation. The guide RNA used for each individualformulation is selected from Table 5. The mRNA can be Trilink mRNA,MS004, or MA004, or any other mRNA.

The targeting lipids of Example 24-A is then successively replaced withtargeting lipids 1012, 1014, 1051 and 1053 (Table 4), where thedistearylglycerol moiety is replaced with a cholesterol moiety. The samenumber of formulations with different ratio of payloads (guide RNA andmRNA) and individual formulation of guide RNA and mRNA are also preparedas described in Example 23 for evaluation. The guide RNA used for eachindividual formulation is selected from Table 5. The mRNA can be TrilinkmRNA, MS004, or MA004, or any other mRNA.

The targeting lipids of Example 24-A are replaced with targeting lipids1062 and 1065 to generate new targeting LNPs for evaluation. In the newformulations thus obtained the distearylglycerol moiety is replaced witha tocopherol moiety.

The targeting lipids of Example 24-A are replaced with targeting lipids1003 to generate new targeting LNPs for evaluation.

Example 25. GalNAc-Lipid Post-Addition Processes with LNPs

Certain LNPs compositions of Tables 6, 8, 9-12 and 7 were formulated andallowed to rest for a range of 1 minute to 120 minutes. The stealthlipid was included in the initial lipid mixture and/or in the dilutionbuffer at a mol % of 0-5 in some instances. In some instances,GalNAc-Lipid 1004, among others, was added in an ethanol/aqueoussolution at a mol % of 0.01-10 following LNP formulation. It was addedin the range of 1 minutes to 120 minutes following LNP formulation andallowed to interact with the LNPs in ethanol/aqueous buffer for 1 minuteto 120 minutes before buffer exchange into formulation buffer. In somecases, the buffer exchange was carried out by PD-10 column, in others itwas dialysis, and in other instances TFF was used. In some cases, TFFwas used to concentrate the LNPs, and in other cases an amiconcentrifugation concentration column was used.

Post addition of GalNAc-Lipid to LNPs is described in Tables 8-13. Datais shown therein.

TABLE 8 Exemplary lipid compositions Receptor targeting conjugate y ofFormula (V) Neutral Stealth or (VI) (e.g., lipid Lipid LNP Lipid ofcompound (e.g., (e.g., PEG- Formu- Table 6, of Table 4), Cholesterol,DSPC) DMG) lation % mol % mol % mol % mol % mol 7-1 50 0 38.5 10 1.5 7-245 0 44 9 2 7-3 50 0 38.5 9 1.5 7-4 49.99 0.01 38.5 10 1.5 7-5 44.990.01 44 9 2 7-6 49.99 0.01 38.5 9 1.5 7-7 49.9 0.1 38.5 10 1.5 7-8 44.90.1 44 9 2 7-9 49.9 0.1 38.5 9 1.5 7-10 49 1 38.5 10 1.5 7-11 44 1 44 92 7-12 49 1 38.5 9 1.5 7-13 45 5 38.5 10 1.5 7-14 40 5 44 9 2 7-15 45 538.5 9 1.5 7-16 47.1 0.5 46.1 4.7 2.1 7-17-1¹ 47.1 0 46.1 4.7 2.1¹7-17-1 was made with GA055 PCSK9 guide RNA and MS004 Cas9 mRNA

TABLE 9 Example of GalNAc-LNP formulation parameters and characteristicsusing 1004 as GalNAc-lipid. 1004 was added to the LNP post formulation.LNP composition (mol %):iLipid (502)/DSPC/Cholesterol/PEG-DMG (507) =47.1:4.7:46.1:2.1. GalNAc- Lipid Buffer Storage 1004 Addition Dilutionexchange condition LNP ID Cargo types mol % of 1004 buffer process (°C.) 7-16 GA055 + 0.5 Post LNP 16% ethanol Dialysis 2-8 MS004 formulationin PBS to PBS 7-16- GA259 + 16% ethanol Dialysis 2-8 A MA002 in PBS toPBS 7-16- GA259 + 5% ethanol Dialysis 2-8 B MA002 in PBS to PBS 7-16-GA259 + Diluted to Dialysis 2-8 C MA002 5% ethanol to PBS in PBS 7-16-GA259 + 16% ethanol Dialysis 2-8 D MA002 in water to PBS 7-16- GA257 +16% ethanol Dialysis E MA004 in water to 50 mM −80 Tris 7-16- GA055 + 5%ethanol TFF to 50 −80 F MS004 in PBS mM Tris 7-16- GA055 + 16% ethanolDialysis 2-8 G MS004 in PBS to 50 mM Tris 7-16- GA257 + 16% ethanol PD10to 2-8 H MA004 in PBS PBS 7-16- GA257 + 16% ethanol TFF to 50 −80 IMA004 in PBS mM Tris 7-16- GA055 + 16% ethanol Dialysis −80 J MS004 inPBS to 50 mM Tris 7-16- GA257 + 16% ethanol TFF to 50 −80 K MA004 inwater mM Tris 7-16- GA097 + 0.5 16% ethanol TFF to 50 −80 L MA004 inwater mM Tris 7-16- GA097 + 1 16% ethanol TFF to 50 −80 M MA004 in PBSmM Tris 7-16- GA256 + 0.5 16% ethanol Dialysis 2-8 N MA004 in PBS to PBS7-16- GA256 + 0.5 16% ethanol Dialysis 2-8 O* MA004 in PBS to PBS 7-16-GA256 + 0.5 In Dilution 16% ethanol Dialysis 2-8 P* MA004 buffer in PBSto PBS *the LNP composition (mol %) is iLipid(502)/DSPC/Cholesterol/PEG-DMG (507) = 55:4.7:38.2:2.1. The ratiosstated herein are based on the ratio of the LNP components before theaddition of GalNAc lipid. Upon the inclusion of the GalNAc lipid, thecomposition shifts slightly.

TABLE 10 Example of GalNAc-LNP formulation parameters andcharacteristics using 1004 as GalNAc-lipid. 1004 was added to the LNPpost formulation. LNP composition (mol %):iLipid(502)/DSPC/Cholesterol/PEG-DMG (507) = 47.1:4.7:46.1:2.1. 7-19 and 7-20carried MA002 (ABE mRNA) and GA259 (ANGPTL3 guide RNA) at 1:1 ratio.7-22 contained MA004 ABE mRNA and GA256 (PCSK9 guide RNA) at 1:1 ratio.All other stated GalNAc-LNPs were carrying MS004 (SpCas9 mRNA) and GA055(PCSK9 gRNA) at 1:1 ratio. Average RNA LNP mol % Addition DilutionBuffer Storage diameter entrapment ID of 1004 of 1004 buffer exchange (°C.) (nm) PDI (%) 7-17-A 2 Post LNP PBS Dialyzed 4-8 84.4 0.0641 97.067-18 1 formulation PBS to PBS 4-8 77.13 0.15 96.55 7-16 0.5 PBS 4-8 77.50.002 97.3 7-19 0.25 PBS 4-8 76.3 0.02 98.5 7-20 0.05 PBS 4-8 77.04 0.0898.74 7-21 0.5 Added after PBS PD10 4-8 93.5 0.03 — buffer exchange toLNP and then buffer exchanged to PBS 7-22 0.5 Added to −80 88.5 0.0792.2 the final thawed LNP 7-23 0.5 Added to 4-8 91.87 0.01 — the thawedfinal LNP 7-24 0.5 Collected in PBS Dialyzed 4-8 81.5 0.0778 97.0 bufferto PBS containing 1004 7-25 0.5 Collected in water Dialyzed 4-8 83 0.12— water to PBS containing 1004

TABLE 11 Example of GalNAc-LNP formulation parameters andcharacteristics using 1004 as GalNAc-lipid. 1004 was added to the LNP atvarious stages of the formulation process. LNP composition (mol%):iLipid (502)/DSPC/Cholesterol (=47.1:4.7:46.1) remained unchangedduring all the examples. All the GalNAc-LNPs were carrying MS004 (SpCas9mRNA) and GA055 (PCSK9 gRNA) at 1:1 ratio, except 7-39, 7-40, and 7-41which were gRNA GA256 and mRNA MA004 at 1:1 ratio; formulation 7-26contains GA257 gRNA and mRNA MA004; formulation 7-29 contains GA259 gRNAand mRNA MA002 1004 in lipid 1004 in Average RNA LNP- 507 excipientDilution buffer# LNP particle entrapment ID (mol %) stream (mol %) (mol%) diameter (nm) PDI (%) 7-26 0 2.1 0 80.9 0.04 85 7-27 0 1.5 0 124 0.01— 7-28 0 1 0 139 0.04 — 7-29 2.1 0.5 0 73.5 0.02 99.0 7-30 2.1 0.25 0.25103 0.04 — 7-31 1.6 0.25 0 93.4 0.074 96.85 7-32 1.1 0.51 0 98.5 0.05196.46 7-33 0 0.91 0 102 0.053 96.81 7-33-A* 2.1 0.5 — 85.2 0.0521 95.57-39 1.1 1.0 0 85.7 0.0382 94.3 7-40 0 1.5 0 118 0.049 93.6 7-41 2.10.25 0.25 73.1 0.00619 97 post addition #Dilution buffer is 16.5%ethanol in PBS. *GalNAc-Lipid 1004 was added using a third port mixing,as reflected in Process 3 of FIG. 9.

TABLE 12 Example of GalNAc-LNP formulation parameters andcharacteristics using various GalNAc-lipids. LNPs 7-34 and 7-35 weremade following the method of LNP 7-16 (Table 9) whereas 7-36 and 7-37were made following method as described to make 7-16-D (Table 9). LNPcomposition (mol %):iLipid (502)/DSPC/Cholesterol/PEG- DMG (507) =47.1/4.7/46.1/2.1. All the GalNAc-LNPs carried GA259 (gRNA) and MA004(mRNA) at 1:1 ratio, except 7-38A was gRNA GA256 and mRNA MA004 GalNAc-Average RNA en- LNP GalNAc- lipid diameter trapment ID lipid mol %addition (nm) PDI (%) 7-34 1053 0.5 Post LNP 76.2  0.13  98.37 7-35 10140.5 formu- 73.5  0.1  99.1  7-36 1043 0.5 lation 79   0.1  97.06 7-371002 0.5 80.55 0.17  98.07 7-38- 1044 0.5 74.1  0.036 96   A

Evaluation of LNP Components Using HPLC

The lipid composition of the LNP was characterized by HPLC methods asseen in FIG. 1A-FIG. 1B. The HPLC methods used were an ion-pairingreverse phase high performance liquid chromatography with evaporativelight scattering detection (IP-RPLC-HPLC-ELSD) to quantify the % of eachlipid in a given sample. Each lipid component was calibrated against astandard curve with sample detection above the limit of quantitation(S/N≥10). FIG. 1A-FIG. 1B display the HPLC chromatogram demonstratingGalNAc-lipid incorporation: (FIG. 1A) reference LNP with no GalNAc-Lipidpresent and (FIG. 1B) LNP constituted with GalNAc-Lipid. The HPLC peakat retention time (RT) 6.721 min labeled as PEG-DMG is PEG-Lipid 507 andthe peak at RT 8.176 min labeled as VL01 is the amino lipid 502 in FIG.1A. The HPLC peak at RT 6.901 min labeled as PEG-DMG is PEG-Lipid 507,the peak at RT 8.402 labeled as VL01 is the amino lipid 502 and the HPLCpeak at RT 10.749 min labeled as GalNAc is 1004 in FIG. 1B.

Example 26. LNPs Constituted with PEG-DSG (508)

The PEG-lipid excipients used in Examples 23, 24 and 25 are replacedwith PEG-DSG (508, Table 7) to obtain targeting and non-targeting LNPsfor further evaluation and to compare delivery efficiency and geneediting in vitro and in vivo under LNP-mediated delivery/uptakeconditions.

Example 27. LNPs Constituted with PEG-Cholesterol (504)

The PEG-DSG used for constituting LNPs in Example 26 is replaced withPEG-Cholesterol (504 and 505, Table 7) to obtain targeting andnon-targeting LNPs for further evaluation and to compare deliveryefficiency and gene editing in vitro and in vivo under LNP-mediateddelivery/uptake conditions.

Example 28. LNPs Constituted with Single mRNA Payload

The payloads in Examples 23-27 are replaced with a single mRNA payload(Molecular Therapy 2018, 26, 1509-1519) to evaluate mRNA expression invitro and in vivo under LNP-mediated delivery/uptake conditions.

Example 29. LNPs Constituted with Single siRNA Payloads

The payload in Examples 23-28 are replaced with an siRNA to evaluatedRNAi-mediated gene silencing. The siRNA used for evaluation is thereported FVII siRNA under LNP-mediated delivery/uptake conditions(Jayaraman et al, Angew. Chem. Int. Ed. 2012, 51, 8529-8533).

Example 30. LNPs Constituted with Antisense Oligonucleotide Payloads

The payload in Examples 23-28 is replaced with antisense oligonucleotideto evaluated antisense effect in vitro and in vivo under LNP-mediateddelivery/uptake conditions (Prakash et al., ACS Chemical Biology, 2013,8(7), 1402-1406).

Example 31. LNPs Constituted with Antimir/Antagomir Payloads

The payload in Examples 23-28 is replaced with a miRNA for miRNAactivity evaluation in vitro and in vivo under LNP-mediateddelivery/uptake conditions (Zhang et al., J. Controlled Release 2013,168, 251-261; Kruetzfeldt et al., Nature 2005, 438, 685-689).

Example 32. LNPs Constituted with microRNA Payload

The payload in Example 26 is replaced with a miRNA for microRNA activityevaluation in vitro and in vivo under LNP-mediated delivery/uptakeconditions (Wang et al., J. Control Release 2013, 28, 690-8).

Example 33. In Vitro Evaluation of LNPs

Gene editing activity of the LNPs obtained from Examples 23-27 areevaluated in hepatocytes (rodent, monkey and human) as described in Finnet al., Cell Reports 2018, 22, 2227-2235.

Editing efficiency of LNPs these LNPS are tested in the presence and inthe absence of serum in the media.

In addition, LNPs obtained Examples 23-27 are tested in the above celllines under serum-free conditions and in the presence or absence ofrecombinant human ApoE (Akinc et al, Mol Ther. 2010, 18, 1357-64).

Gene editing activity of all these LNPs are also evaluated in ASGPR,LDLr and ApoE knockout hepatocytes (human, monkey and rodent) under allconditions described above.

Example 34. Evaluation of GalNAc-LNP Binding to ASGPR

ASGPR biding of all ASGPR targeting LNPs obtained from Examples 23-31 ismeasured as described in Mol Ther. 2010, 18, 1357-64.

Example 35. GalNAc-Lipid Inclusion after LNP Formation

The LNPs were formulated and allowed to rest for a range of 1 min to 120min. The stealth lipid was included in the initial lipid mixture and/orin the dilution buffer at a mol % of 0-5 in some instances. The LNPswere buffer exchanged and concentrated, in some cases using TFF toconcentrate, and in other cases an amicon centrifugation concentrationcolumn was used. In some instances, the buffer exchange was carried outby PD-10 column, in others it was dialysis, and in other instances TFFwas used. GalNAc-Lipid 1004 was/is then added in an ethanol/aqueoussolution at a mol % of 0.01-10, in the range of 1 to 120 minutesfollowing LNP concentration, and allowed to interact with the LNPs.

In another formulation approach, LNPs are formulated and allowed to restfor a range of 1 minute to 120 minutes. The stealth lipid may beincluded in the initial lipid mixture and/or in the dilution buffer at amol % of 0-5 in some instances. The LNPs are diluted in dilution bufferin a range of 1 to 1000% of the initial volume. GalNAc-Lipid 1004 isthen added in an ethanol/aqueous solution at a mol % of 0.01-10, in therange of 1 minute to 120 minutes following LNP formulation, and allowedto interact with the LNPs in ethanol/aqueous buffer for 1 minute to 120minutes before buffer exchange into final formulation buffer at a pH 7or 8.

The LNPs are formulated as described above and buffer exchanged intofinal formulation buffer. The stealth lipid was included in the initiallipid mixture and/or in the dilution buffer at a mol % of 0-5 in someinstances. GalNAc-Lipid 1004 in solution is then added at a mol % in arange of 0.01-10, allowed to interact for a range of 1 to 120 minutes,and then the solution is buffer exchanged into the final formulationbuffer.

In other embodiments, the LNPs are formulated as described above andbuffer exchanged into the final formulation buffer. The stealth lipidwas included in the initial lipid mixture and/or in the dilution bufferat a mol % of 0-5 in some instances. GalNAc-Lipid 1004 in solution isthen added at a mol % in a range of 0.01-10, allowed to interact for arange of 1 to 120 minutes.

In some embodiments, the LNPs are collected post-formulation directlyinto a dilution buffer containing GalNAc-Lipid 1004 in solution at a mol% in the range of 0.01-10. The stealth lipid may be included in theinitial lipid mixture and/or in the dilution buffer at a mol % of 0-5 insome instances. The GalNAc-Lipid 1004 incorporates into the LNPs insolution and the solution is buffer exchanged into the final formulationbuffer following a period of 1 to 120 minutes.

LNPs were/are also formulated and buffer exchanged into the finalformulation buffer. Cryoprotectant is then added to the LNPs thus formedto store at −80° C. as described in Example 24. The LNPs devoid ofcryoprotectant are stored at 2-8° C. and the final formulation thatcontains the cryoprotectant are stored at −80° C. The frozen LNPs arethen thawed at room temperature. GalNAc-Lipid 1004 in an ethanol/aqueoussolution is then added to the thawed LNPs at a mol % of 0.01-10. In someinstances, the LNPs are then buffer exchanged into the final formulationbuffer. In other instances, they are not buffer exchanged followingGalNAc-Lipid addition.

In some instances, buffer exchange was performed through a PD-10desalting column (column packed with Sephadex to separate high from lowmolecular weight compounds by desalting and buffer exchange), dialysis,or Tangential Flow Filtration (TFF). In some instances, the LNPs arestored at 2-8° C. or −80° C. following GalNAc-Lipid addition and bufferexchange.

GalNAc-LNPs are then constituted by replacing the GalNAc-Lipid 1004 withother GalNAc-Lipids from Table 4.

Example 36. GalNAc-Lipid Inclusion in Pre-Formulation Lipid Mixture toObtain GalNAc-LNP

The exemplary GalNAc-Lipid 1004 from Table 4 was included in the initiallipid mixture (including but not limited to: ionizable lipid, stealthlipid, helper lipid, etc.) at a mol % of 0.01-10, pre-formulation ofLNPs. The stealth lipid was included at a mol % of 0-5 in someinstances. LNPs were formulated and buffer exchanged in a range of 1minute to 1 day following formulation and were stored as described inExample 24.

The exemplary GalNAc-Lipid 1004 from Table 4, in some instances, isincluded in the initial lipid mixture (including but not limited to:ionizable lipid, stealth lipid, helper lipid, etc.) at a mol % of0.01-10, pre-formulation of LNPs. The stealth lipid was included at amol % of 0-5 in some instances. The LNPs were formulated and collecteddirectly into a solution containing 0.01-10 mol % of GalNAc-Lipid 1004.The LNPs are allowed to rest for 1 to 120 min before being bufferexchanged into final formulation buffer for storing at 2-8° C. and/orfor storing at −80° C., as described in Example 24.

GalNAc-Lipid 1004, in some instances, was included in the initial lipidmixture (including but not limited to: ionizable lipid, stealth lipid,helper lipid, etc.) at a mol % of 0.01-10, pre-formulation of LNPs. Thestealth lipid was included at a mol % of 0-5 in some instances. The LNPswere formulated and allowed to rest for 1 min to 120 min. GalNAc-Lipidin an ethanol/aqueous solution was/is then added to the LNPs at a mol %of 0.01-10 and the mixture is allowed to rest for a further 1 min to 120min. The LNPs are then buffer exchanged into final formulation bufferfor storing at 2-8° C. and/or for storing at −80° C., as described inExample 24.

GalNAc-Lipid 1004, in some instances, is included in the initial mixtureof lipids (including but not limited to: ionizable lipid, stealth lipid,helper lipid, etc.) at a mol % of 0.01-10, pre-formulation of LNPs. Thestealth lipid may be included at a mol % of 0-5 in some instances. TheLNPs are formulated and then buffer exchanged into storage buffer forstoring at 2-8° C. and/or for storing at −80° C., as described inExample 24, and stored at 2-8° C. and/or −80° C. The LNPs are thenthawed at room temperature. GalNAc-Lipid 1004 in an ethanol/aqueoussolution is then added to the LNPs at a mol % of 0.01-10, and themixture is allowed to rest for a further 1 min to 120 min.

LNPs were stored as described in Example 24. In some instances, bufferexchange is/was performed through a PD-10 column, dialysis, orTangential Flow Filtration (TFF). In some instances, the LNPs are/werestored at 2-8° C. or −80° C. following GalNAc-Lipid addition andappropriate buffer exchange depending on the desired storage conditions.

GalNAc-LNPs are then constituted by replacing the GalNAc-Lipid 1004 withother GalNAc-Lipids from Table 4.

Example 37. In Line Dilution of LNPs with GalNAc to ConstituteGalNAc-LNP

GalNAc-LNPs were formulated using an in-line (third channel) dilutionmethod. One channel/line is the lipid mixture (including but not limitedto: ionizable lipid, stealth lipid, helper lipid, etc.). The otherchannel/line contains cargo in an aqueous solution (including but notlimited to: guide RNA and mRNA). The third channel/line contains thedesired GalNAc-Lipid 1004 in an ethanol/aqueous solution such that thefinal mol % in the LNPs is in the range of 0.01-10. The LNPs wereallowed to rest for 1 min to 120 min before being buffer exchanged intothe final formulation buffer and stored as described in Example 24.

In other instances, GalNAc-LNPs are formulated using an in-line (thirdchannel) dilution method. One channel/line is the lipid mixture(including but not limited to: ionizable lipid, stealth lipid, helperlipid, etc.). The other channel/line contains cargo in an aqueoussolution (including but not limited to: guide RNA and mRNA). The thirdchannel/line contains the desired GalNAc-Lipid 1004 in anethanol/aqueous solution such that the final mol % in the LNPs is in therange of 0.01-10%. LNPs are then collected directly into a solutioncontaining 0.01-10 mol % of the same GalNAc-Lipid. The LNPs are allowedto rest for 1 min to 120 min before being buffer exchanged into thefinal formulation buffer for storing at 2-8° C. and/or for storing at−80° C., as described in Example 24.

In other instances, LNPs are formulated using an in-line (third channel)dilution method. One channel/line is the lipid mixture (including butnot limited to: ionizable lipid, stealth lipid, helper lipid, etc.). Theother channel/line contains cargo in an aqueous solution (including butnot limited to: guide RNA and mRNA). The third channel/line contains thedesired GalNAc-Lipid from Table 4 in an ethanol/aqueous solution suchthat the final mol % in the LNPs is in the range of 0.01-10. The LNPsare formulated and allowed to rest for 1 min to 120 min. GalNAc-Lipid1004 in an ethanol/aqueous solution is then added to the LNPs at a mol %of 0.01-10 and the mixture is allowed to rest for a further 1 to 120min. The LNPs are then buffer exchanged into the final formulationbuffer for storing at 2-8° C. and/or for storing at −80° C., asdescribed in Example 24.

LNPs were/are stored as described in Example 24. In some instances,buffer exchange is/was performed through using a PD-10 column, bydialysis, or by Tangential Flow Filtration (TFF). In some instances, theLNPs are stored at 2-8° C. or −80° C. following GalNAc-Lipid additionand appropriate storage buffer exchange.

GalNAc-LNPs are then constituted by replacing the GalNAc-Lipid 1004 withother GalNAc-Lipids from Table 4.

Example 38. In Vivo Gene Editing Evaluation of LNPs

Gene editing activity of a number of formulated LNPs from Examples 23-27were evaluated in wild-type, and LDLr and ApoE knockout rodent modelsand in non-human primates.

Mice were treated in accordance with institutional ethical guidelines ofanimal care, handling, and termination. Mice were kept in apathogen-free facility, with free access to standard chow and water.Mice were dosed with test articles or PBS as a vehicle by retro-orbitalroute according to their bodyweight. Relevant tissues were collectedpost dosed fifth or sixth day. Genomic DNA was extracted, and percentediting of target sequence was evaluated by next-generation sequencingto determine editing efficiency and the results are depicted in FIGS.3-8.

FIG. 2. illustrates PCSK9 editing efficiency in primary humanhepatocytes in vitro following transfection with 7-17, 7-16-L, and7-16-M for three days before harvesting for NGS analysis as described inExample 33. LNPs were dosed in a dose response ranging from 312.5 ng/mLLNPs concentration to 2500 ng/mL LNPs concentration.

FIG. 3 illustrates PCSK9 gene editing in wild type (grp1 and 2), LDLRknockout (LDLR−/−, grp 3 and 4) and ApoE knockout (ApoE−/−, grp 5 and 6)mice (n=5) liver at 1 mg/kg dose, after retro-orbital administration ofLNPs 7-17-1 and 7-16 carrying SpCas9 mRNA (MS004) and PCSK9 gRNA (GA055)at 1:1 ratio. 7-17-1 is the reference LNP and 7-16 is the GalNAc-LNPwith 1004 as GalNAc-Lipid. grp 1, 3 and 5 were treated with 7-17-1, andgrp 2, 4 and 6 were treated with 7-16. LNP IDs are given in Table 8 and9. The reference LNP 7-17-1 that lacks the GalNAc-Lipid produced low orvery poor editing in LDLR−/− and ApoE−/− mice.

FIG. 4 illustrates gene editing in LDLR−/− mice liver afterretro-orbital administration of GalNAc-LNPs compositions herein carryingABE mRNA MA002 and gRNA GA259 at 1:1 ratio. 7-16-A, 7-16-B and 7-16-Cwere prepared as described in Table 9 with ANGPTL3 guide GA259 and ABEmRNA MA002; 7-19 and 7-20 were prepared as described in Table 10 withANGPTL3 guide GA259 and ABE mRNA MA002; 7-29 was prepared as describedin Table 11 with ANGPTL3 guide GA259 and ABE mRNA MA002; and 7-34 and7-35 were prepared as described in Table 12 with ANGPTL3 guide GA259 andABE mRNA MA002. The data highlights the dose response of 7-16-A as wellas the effect of formulation process of GalNAc-LNPs (7-16-B, 7-16-C,7-29) on hepatic gene editing activity. 7-19 and 7-20 illustrates theeffect of GalNAc-Lipid (1004) mol % titration on hepatic gene editing invivo. 7-34 and 7-35 (Table 4) have cholesterol as a lipid anchor. Theediting data of 7-29 and 7-35 conclude that the lipid anchor impacts invivo efficacy of GalNAc-LNP.

FIG. 5 illustrates the PCSK9 gene editing in wild type (grp1, 2 and 3)and LDLR−/− (grp 4, 5, 6 and 7) mice (n=5) liver after retro-orbitaladministration of LNPs carrying ABE mRNA (MA004) and PCSK9 gRNA (GA257)at 1:1 ratio. 7-16-I was administered to grp 1, 4, and 5; grps 2 and 6were treated with 7-16-H at 0.25 mg/mL total RNA dose; 7-26 wasadministered to groups 3 and 7 at 0.25 mg/mL. Grp 1, 2, 4, and 6 weredosed at 0.25 mg/kg; and grp 5 was dosed at 0.125 mg/kg. LNP IDs andformulation information are given in Table 9 and Table 11. Formulation7-16-H and 7-16-I compared editing efficiency of the same formulationwhen buffer exchanged by PD-10 column and stored at 2-8° C., and bufferexchange via TFF and stored at −80° C. respectively. In formulation 7-26(grp3 wild-type; grp7 LDLR−/− at 0.25 mg/kg) the PEG-Lipid 507 wascompletely replaced with GalNAc-Lipid 1004. Grp 1 (7-16-I), grp 2(7-16-H), grp 4 (7-16-I) and grp 6 (7-16-H) illustrate the effect ofbuffer exchange conditions of same GalNAc-LNPs lipid composition (Table9) on hepatic gene editing in WT and LDLR−/− mice respectively. Grps 4and 5 were treated with formulation 7-16-I at 0.25 and 0.125 mg/kg doserespectively to show the dose response effect on hepatic gene activityin LDLR−/− mice.

FIG. 6 illustrates PCSK9 gene editing in wild type female mice (n=5)hepatocytes after retro-orbital administration of LNPs 7-16-J and 7-16-E(Table 9). 7-16-J was constituted with SpCas9 mRNA (MS004) and PCSK9gRNA (GA055) and 7-16-E with ABE mRNA (MA004) and PCSK9 (GA257) gRNA at1:1 ratio. 7-16-J and 7-16-E were dosed at 0.25 mg/kg and at 0.5 mg/kg,respectively.

FIG. 7 illustrates the PCSK9 gene editing in wild type female mice (n=5)hepatocytes after retro-orbital administration of LNPs 7-16-I and 7-16-Kat 0.25 mg/kg. Both LNPs contain ABE mRNA MA004 and PCSK9 gRNA GA257.Both formulations contain same excipient and 1004 mol %. For thepreparation of 7-16-I and 7-16-K the LNPs after inline mixing werecollected in PBS containing 16% ethanol and water containing 16%ethanol, respectively. LNP IDs and formulation information are given inTable 9.

FIG. 8 illustrates PCSK9 editing in LDLR−/− female mice (n=5)hepatocytes after retro-orbital administration of LNPs carrying Cas9mRNA and gRNA at 0.5 mg/kg dose. 7-17-1 (Table 8), 7-16 (Table 9), 7-18(Table 10), 7-17-A (Table 10), 7-24 (Table 10), 7-33-A (Table 11), 7-23(Table 10) and 7-33 (Table 11) constituted with spCas9 mRNA and gRNA.7-17-1 (Table 8), 7-16 (table 9), 7-18 (Table 10) and 7-17-A (Table 10)shows the effect of GalNAc-Lipid (1004) mol % (0, 0.5, 1.0 and 2.0 mol%, respectively) titration on gene editing activity in LDLR−/−micehepatocytes. All three formulation were prepared by following Process 1in FIG. 9. The effect of various formulation processes on gene editingpotency is being tested by 7-24, 7-33-A, 7-23 and 7-33 GalNAc-LNPs.Formulation 7-17-1 is the control LNP that lacks the GalNAc-Lipid 1004.7-24 contains 0.5 mol % of 1004 where the GalNAc-Lipid was added to thecollection buffer (Process 2, FIG. 9). 7-33-A was prepared byintroducing the GalNAc-Lipid 1004 through third port/in-line mixing intothe mixing chamber (Process 3, FIG. 9). 7-23 the GalNAc-Lipid was addedto pre-formed LNP stored at −80° C., after thawing prior toretro-orbital administration to the mice. 7-33 was prepared by premixingGalNAc-Lipid 1004 with other lipid excipients (Process 4, FIG. 9). 7-34and 7-35 (Table 12) were constituted with ABE mRNA and ANGPTL3 gRNA at1:1 ratio where the GalNAc-Lipid 1004 was replaced with 1053 and 1014respectively (post insertion 0.5 mol %), and demonstrates thatGalNAc-LNP containing 1004 is capable of being a more efficaciousdesign.

FIG. 15 illustrates the PCSK9 gene editing in LDLR−/− female mice (n=5)hepatocytes after retro-orbital administration of LNPs 7-16-N and 7-38-Aat 0.25 mg/kg. 7-16-N includes 1004 and is described in Table 9, and7-38-A includes 1044 and is described in Table 12. 7-16-N and 7-38-Acontain PCSK9 guide RNA GA256 and ABE mRNA MA004. 1004 shows higherefficacy for the same method of formulation as compared to 1044. Theywere both made according to Process 1 in FIG. 9 and Protocol 1 in FIG.10.

LNPs were also dosed to cynomolgus non-human primates at 1 mg/kg dose.LNPs were given via IV infusion over 1 hour. LNPs were prepared with LNPIDs given in Table 5 and/or 7, according to GalNAc-Lipid additionmethods described in Examples 24, and shown in Table 13. LNPs hadpreviously been stored at −80° C. as described in Example 24. LNP 7-17served as control, while LNPs 7-16-L and 7-16-M had GalNAc-Lipid 1004 inthe formulation. Primates were sacrificed on Day 1 or Day 14 followingdosing. The end points included but were not limited to: LDL-c levels inblood, % editing of target gene in liver hepatocytes, PCSK9 proteinlevels in blood, ANGPTL3 protein levels in blood, among other lipidparameters.

These formulations were then incubated with human and monkey primaryhepatocytes as described in Example 33 and the results are summarized inFIG. 2. FIG. 2 illustrates in vitro PCSK9 gene editing efficiency inprimary human hepatocytes of LNP formulations 7-17, 7-16-L and 7-16-M.

TABLE 13 LNPs were prepared as described in Examples 23 and 24, and werestored at −80° C. as described in Example 24. They were then tested innon-human primates. LNPs were administered via IV infusion at 1 mg/kgdose mol % Average Dose RNA en- Dose Dose LNP- gRNA + No. of GalNAc- LNPsize (mg/ trapment Volume Route/ ID mRNA Animals Lipid 1004 (nm) PDI kg)(%) (mL/kg) Regimen 7-17 MA004 + 5 0 91 0.05 1 96.2 6 IV inf., GA097 1 hD0 7-16- MA004 + 5 0.5 73.1 0.06 1 97.1 6 IV inf., L GA097 1 h D0 7-16-MA004 + 3 1 79 0.09 1 96.5 6 IV inf., M GA097 1 h D0

Genomic DNA Isolation

Genomic DNA was isolated from approximately 20 μL of whole mouse liverlysate using a bead-based extraction kit, MagMAX-96 DNA Multi-Sample Kit(Thermo-Fisher Scientific) on the KingFisher Flex automated extractioninstrument (Thermo-Fisher Scientific) according to the manufacturer'sprotocols. Mouse whole liver was lysed using the FastPrep-24 system (MPBio) according the to manufacturer's protocol. Livers were loaded into 2mL lysing matrix tubes (MP Bio) with 0.5 mL of PBS. Extracted genomicDNA was stored at 4° C. until further use or at −80° C. for long termstorage.

Next Generation Sequencing (NGS) and Analysis of Editing Efficiency

Next generation sequencing (NGS), or deep sequencing, was performed onthe region of interest to determine the extent of gene editing. Sampleswere prepared using the Nextera XT DNA library preparation kit(Illumina) according to the manufacturer's protocol. Briefly, two roundsof PCR were performed first to amplify the region of interest and secondto add DNA sequences required for deep sequencing and sampleidentification to the initial product. The final amplicon was sequencedon the Illumina MiSeq instrument according to the manufacturer'sprotocol. Paired-end reads were analyzed with the CRISPResso2 pipeline(see Clement, K., Rees, H., Canver, M. C. et al. CRISPResso2 providesaccurate and rapid genome editing sequence analysis. Nat Biotechnol 37,224-226 (2019). Briefly, low-quality reads were filtered out, adaptersequences were trimmed from the reads, and the paired-end reads weremerged and aligned to the amplicon sequence. The editing percentage wascalculated as the number of reads supporting an insertion or a deletion,over the total number of aligned reads. For Cas9, the editing percentagewas calculated as the number of reads supporting an insertion or adeletion, over the total number of aligned reads.

Example 39. GalNAc-LNPs Constituted from GalNAc-Lipid 1076

The desired GalNAc-LNPs are constituted by replacing GalNAc-Lipid ofExamples 25-38 with GalNAc-Lipid 1076.

Example 40. GalNAc-LNPs Constituted from GalNAc-Lipid 1079

The desired GalNAc-LNPs are constituted by replacing GalNAc-Lipid ofExamples 25-38 with GalNAc-Lipid 1079.

FIGS. 9-14 as previously noted are illustrations of representativemanufacturing processes for GalNAc-LNPs disclosed herein. Illustrated inFIG. 9 are Processes 1-4. Process 1 depicts a method of preparation ofGalNAc-LNPs, aspects of which are described more completely in Sato, etal J. Controlled Release, 2017, 266, 216-225. Process 2 illustrates amethod for the creation of LNPs, whereby nucleic acids in an aqueousbuffer are sent into a mixer through one (or more) channels and lipidcomponents (including but not limited to: amino lipid, helper lipid,structural lipid/sterol, and stealth lipid) are sent into the mixerthrough a separate one or more channels. It is to be understood that thetwo streams of the RNA payload and LNP excipients are entering the mixerthrough separate channels and mixing inside the mixer; they do notcontact each other before entering the mixer. The transiently formed LNPin the mixing chamber is then collected in dilution buffer containingGalNAc-Lipid at the desired mol %. The GalNAc-LNPs then enter holdingtime before proceeding through buffer exchange. Process 3 illustrates anin-line mixing method for the preparation of GalNAc-LNPs, wherebynucleic acids in an aqueous buffer are sent into an in-line mixerthrough one (or more) channels and lipid components (including but notlimited to: amino lipid, helper lipid, structural lipid, and stealthlipid) are sent into the mixer through a separate one or more channels.It is to be understood that the two streams of the RNA payload and LNPexcipients are entering the mixer through separate channels and mixinginside the mixer; they do not contact each other before entering themixer. In some instances, the mixer is a T mixer, in other instances, itis a cross mixer. The solution then travels through a very shortdistance of tubing to immediately and successively enter another in-linemixer, where the output of the first mixer is in-line mixed withdilution buffer containing GalNAc-Lipid, such that a final desired mol %target compared to the other lipids is achieved. The distance betweenthe two mixers is very short and so the two successive mixing events maybe understood as to be almost instantaneous. The GalNAc-LNPs then entera hold time before proceeding to buffer exchange. Process 4 illustratesa method for the preparation of GalNAc-LNPs, whereby nucleic acids in anaqueous buffer are sent into an in-line mixer through one (or more)channels and lipid components (including but not limited to: aminolipid, helper lipid, structural lipid, stealth lipid, and at least aportion or the desired mol % of the GalNAc-Lipid) are sent into themixer through a separate one or more channels. It is to be understoodthat the two streams of the RNA payload and LNP excipients are enteringthe mixer through separate channels and mixing inside the mixer; they donot contact each other before entering the mixer. The GalNAc-LNPs thenare mixed with dilution buffer, and proceed to hold time and bufferexchange. In Process 4, the GalNAc-Lipid is included either entirely orat least partially in the stream containing the lipid excipients thatenters the mixer. The remainder of the GalNAc-Lipid (such that a desiredtarget final mol % as compared to other lipids is achieved), if any, maythen be included through a successive in-line mixer as described inProcess 3, or may be added at a later point in the process.

As illustrated in FIGS. 10-14, specific protocols may be employed witheach of the foregoing processes that further detail specific aspects ofthose processes. FIG. 10 illustrates Protocols 1-3 that can be used inconnection with the processes of FIG. 9 to generate GalNAc-LNPs, wherebynucleic acids in an aqueous buffer are/were sent into a mixer throughone (or more) channels and lipid components (including but not limitedto: amino lipid, helper lipid, structural or sterol lipid, and stealthlipid) are/were sent into the mixer through a separate one or morechannels. It is to be understood that the two streams of the RNA payloadand LNP excipients are/were entering the mixer through separate channelsand mixing inside the mixer; they do not contact each other beforeentering the mixer. The mixed solution then exits the mixer. Protocols1, 2, and 3 are versions of Process 1 that differ only in at what stagethe GalNAc-Lipid is added via post-addition.

FIG. 11 illustrates Protocols 4-6 that can be used in connection withthe processes of FIG. 9 to generate GalNAc-LNPs, whereby nucleic acidsin an aqueous buffer are sent into a mixer through one (or more)channels and lipid components (including but not limited to: aminolipid, helper lipid, structural or sterol lipid, and stealth lipid) aresent into the mixer through a separate one or more channels. It is to beunderstood that the two streams of the RNA payload and LNP excipientsare entering the mixer through separate channels and mixing inside themixer; they do not contact each other before entering the mixer. Themixed solution then exits the mixer. Protocol 4 is a version of Process1 in FIG. 9 where GalNAc-Lipid is added after concentration. Protocol 5utilizes Process 2 in FIG. 9, where GalNAc-Lipid is contained in thestatic dilution buffer in the collection vessel. Protocol 6 utilizesProcess 1 where GalNAc-Lipid is added after concentration, filtration,and storage.

FIG. 12 illustrates Protocols 7-9 that can be used in connection withthe processes of FIG. 9 to generate GalNAc-LNPs, whereby nucleic acidsin an aqueous buffer are sent into a mixer through one (or more)channels and lipid components (including but not limited to: aminolipid, helper lipid, structural or sterol lipid, stealth lipid, and atleast a portion of the GalNAc-Lipid) are sent into the mixer through aseparate one or more channels. It is to be understood that the twostreams of the RNA payload and LNP excipients are entering the mixerthrough separate channels and mixing inside the mixer; they do notcontact each other before entering the mixer. The mixed solution thenexits the mixer. The diagram indicates that the stream exiting themixture enters into dilution buffer. This is understood to mean thestream either contacts a static dilution buffer in a collection vesselor enters a very short length of tubing that conveys the stream into asuccessive in-line mixer in which the stream from the first mixer isin-line mixed with dilution buffer in this successive mixer. Protocol 7utilizes Process 4 from FIG. 9 to generate GalNAc-LNPs. Here,GalNAc-Lipid is entirely included in the mix of other lipids enteringthe mixer. The separate nucleic acid aqueous stream and lipid stream mixinside the mixer before exiting and contacting dilution buffer in one ofthe ways described above. Protocol 8 utilizes a version of Process 4from FIG. 9 to generate GalNAc-LNPs. Here, GalNAc-Lipid is included inthe mix of other lipids entering the mixer, as well as in the dilutionbuffer, which may be included in the protocol in either of the two waysdescribed above. Protocol 9 utilizes a version of Process 4 from FIG. 9to generate GalNAc-LNPs. Here, GalNAc-Lipid is included in the mix ofother lipids entering the mixer, and is added to the GalNAc-LNPs at alater point in the process—in this case after dilution buffer isintroduced to the GalNAc-LNPs in either way as described above and theycomplete a hold time, before completing another hold time followingGalNAc-Lipid addition and being buffer exchanged.

FIG. 13 illustrates Protocols 10 and 11 that can be used in connectionwith the processes of FIG. 9 to generate GalNAc-LNPs, whereby nucleicacids in an aqueous buffer are sent into a mixer through one (or more)channels and lipid components (including but not limited to: aminolipid, helper lipid, structural or sterol lipid, stealth lipid, and atleast a portion of the GalNAc-Lipid) are sent into the mixer through aseparate one or more channels. It is to be understood that the twostreams of the RNA payload and LNP excipients are entering the mixerthrough separate channels and mixing inside the mixer; they do notcontact each other before entering the mixer. The mixed solution thenexits the mixer. The diagram indicates that the stream exiting themixture enters into dilution buffer. This is understood to mean thestream either contacts a static dilution buffer in a collection vesselor enters a very short length of tubing that conveys the stream into asuccessive in-line mixer in which the stream from the first mixer isin-line mixed with dilution buffer in this successive mixer. Protocol 10utilizes a version of Process 4 from FIG. 9 to generate GalNAc-LNPs.Here, GalNAc-Lipid is included in the mix of other lipids entering themixer, and is added to the GalNAc-LNPs at a later point in theprocess—in this case after buffer exchange. Protocol 11 utilizes aversion of Process 4 from FIG. 9 to generate GalNAc-LNPs. Here,GalNAc-Lipid is included in the mix of other lipids entering the mixer,and is also present, possibly with none or a portion of other lipidcomponents, in the dilution buffer. The dilution buffer is understood tocontact GalNAc-LNPs in either of the ways described above.

FIG. 14 illustrates Protocols 12 and 13 that can be used in connectionwith the processes of FIG. 9 to generate GalNAc-LNPs, whereby nucleicacids in an aqueous buffer are sent into a mixer through one (or more)channels and lipid components (including but not limited to: aminolipid, helper lipid, structural or sterol lipid, stealth lipid, and atleast a portion of the GalNAc-Lipid) are sent into the mixer through aseparate one or more channels. It is to be understood that the twostreams of the RNA payload and LNP excipients are entering the mixerthrough separate channels and mixing inside the mixer; they do notcontact each other before entering the mixer. The mixed solution thenexits the mixer and enters a very short length of tubing that conveysthe stream into a successive in-line mixer in which the stream from thefirst mixer is in-line mixed with dilution buffer that containsGalNAc-Lipid in this successive mixer. Protocol 12 utilizes Process 3 inFIG. 9 to generate GalNAc-LNPs. Here, some or none of the GalNAc-Lipidis included in the stream with the other lipids that enters the first Tmixer. It is to be understood that the protocol depicted in Protocol 12can be carried out with or without GalNAc-Lipid in the stream with theother lipids that enters the first mixer. The stream that exits thefirst T mixer is then conveyed via a very short length of tubing intoanother successive T mixer, where it is mixed with dilution buffercontaining GalNAc-Lipid. Protocol 13 utilizes Process 3 in FIG. 9 togenerate GalNAc-LNPs. Here, some or none of the GalNAc-Lipid is includedin the stream with the other lipids that enters the first cross mixer.It is to be understood that the protocol depicted in Protocol 13 can becarried out with or without GalNAc-Lipid in the stream with the otherlipids that enters the first mixer. The stream that exits the firstcross mixer is then conveyed via a very short length of tubing into asuccessive T mixer, where it is in-line mixed with dilution buffercontaining GalNAc-Lipid.

Example 41. Preparation of Exemplary RNA GalNAc Conjugate

The RNA-GalNAc conjugate 2-1, for example, is prepared by thehybridization of a 13-mer oligo(2′-O-methoxyuridine) with a covalentlyconjugated GalNAc ligand at the 5′-end of the sequence (SEQ ID No 8) tothe poly(A) tail at the 3′-end of the RNA (SEQ ID No 7, Table 2). Inconjugate 2-2 the GalNAc ligand is covalently linked to the 3′-end ofthe oligo(2′-O-methoxyuridine) (SEQ ID No 10) that hybridizes with thepoly(A) of the RNA (SEQ ID No 8). In RNA-GalNAc conjugate 2-3, thepoly(A) tail of SEQ ID No 11 is hybridized with aoligo(2′-O-methoxyuridine) (SEQ ID No 12) carrying the GalNAc ligand atboth end of the oligonucleotide. For the preparation of the RNA-GalNAcconjugates 2-4, 2-5 and 2-6, the oligo(2′-O-methoxyuridine) length isincreased to 24-mer. The RNA-GalNAc congates 2-7 to 2-30 are prepared bypartially or completely substituting 2′-O-methoxyuridine (u) either withuridine (U) or thymidine (T) as described in the Table 2. The GalNAcconjugated oligonucleoitdes 8-10 and 12-36 are prepared using the GalNAcligand monomers 37 and 38 (Scheme 3, for example) under solid phaseoligonucleotide synthesis and deprotection as described in J. Am. Chem.Soc. 2014, 136, 16958-16961.

All patents and publications mentioned in this specification are hereinincorporated by reference to the same extent as if each independentpatent and publication was specifically and individually indicated to beincorporated by reference.

OTHER EMBODIMENTS

From the foregoing description, it will be apparent that variations andmodifications may be made to the disclosure described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment, anyportion of the embodiment, or in combination with any other embodimentsor any portion thereof.

As is set forth herein, it will be appreciated that the disclosurecomprises specific embodiments and examples of targeting moietystructures, GalNac conjugate coupling sequences, guide RNA Galnacconjugate designs, GalNac conjugates structures including linkers andother components thereof, GalNac conjugated lipids, gRNA designs andmodifications thereto, lipid compositions, lipid excipient formulations,lipid nanoparticle compositions with and without active agent payloadsincluding but no limited to RNA, lipid nanoparticles comprised ofGalNac-lipids that act as ligands to facilitate transfection andefficacy of active agents in receptor deficient cells and mammals; andspecific examples and embodiments describing the synthesis, manufacture,use, and efficacy of the foregoing individually and in combinationincluding as pharmaceutical compositions for treating disease and for invivo and in vitro delivery of active agents to mammalian cells underconditions where there is an absence of ApoE and/or to such mammaliancells that are deficient in LDL receptors.

While specific examples and numerous embodiments have been provided toillustrate aspects and combinations of aspects of the foregoing, itshould be appreciated and understood that any aspect, or combinationthereof, of an exemplary or disclosed embodiment may be excludedtherefrom to constitute another embodiment without limitation and thatit is contemplated that any such embodiment can constitute a separateand independent claim. Similarly, it should be appreciated andunderstood that any aspect or combination of aspects of one or moreembodiments may also be included or combined with any aspect orcombination of aspects of of one or more embodiments and that it iscontemplated herein that all such combinations thereof fall within thescope of this disclosure and can be presented as separate andindependent claims without limitation. Accordingly, it should beappreciated that any feature presented in one claim may be included inanother claim; any feature presented in one claim may be removed fromthe claim to constitute a claim without that feature; and any featurepresented in one claim may be combined with any feature in anotherclaim, each of which is contemplated herein. The following enumeratedclauses are further illustrative examples of aspects and combination ofaspects of the foregoing embodiments and examples:

-   1. A receptor targeting conjugate, comprising a compound of Formula    (V):

-   -   wherein,    -   A is a receptor targeting moiety;    -   each L¹, L², L³, L⁴, L⁵, L⁶, L⁷, L⁸, L⁹, L¹⁰, and L¹², is        independently substituted or unsubstituted C₁-C₁₂ alkylene,        substituted or unsubstituted C₁-C₁₂ heteroalkylene, substituted        or unsubstituted C₂-C₁₂ alkenylene, substituted or unsubstituted        C₂-C₁₂ alkynylene, —(CH₂CH₂O)_(m)—, —(OCH₂CH₂)_(m)—, —O—, —S—,        —S(═O)—, —S(═O)₂—, —S(═O)(═NR¹)—, —C(═O)—, —C(═N—OR¹)—,        —C(═O)O—, —OC(═O)—, —C(═O)C(═O)—, —C(═O)NR¹—, —NR¹C(═O)—,        —OC(═O)NR¹—, —NR¹C(═O)O—, —NR¹C(═O)NR¹—, —C(═O)NR¹C(═O)—,        —S(═O)₂NR¹—, —NR¹S(═O)₂—, —NR¹—, or —N(OR¹)—;    -   L¹¹ is —(CH₂CH₂O)_(n)— or —(OCH₂CH₂)_(n)—;    -   each R¹ is independently H or substituted or unsubstituted        C₁-C₆alkyl;    -   R is a lipophilic organic residue;    -   m is an integer selected from 1 to 10; and    -   n is an integer selected from 1 to 200.

-   2. The receptor targeting conjugate of clause 1, wherein each L¹,    L⁴, and L⁷ is independently substituted or unsubstituted C₁-C₁₂    alkylene.

-   3. The receptor targeting conjugate of clause 2, wherein each L¹,    L⁴, and L⁷ is independently substituted or unsubstituted C₂-C₆    alkylene.

-   4. The receptor targeting conjugate of clause 2, wherein each L¹,    L⁴, and L⁷ is C₄ alkylene.

-   5. The receptor targeting conjugate of any one of clauses 1-4,    wherein each L², L⁵, and L⁸ is independently —C(═O)NR¹—, —NR¹C(═O)—,    —OC(═O)NR¹—, —NR¹C(═O)O—, —NR¹C(═O)NR¹—, or —C(═O)NR¹C(═O)—.

-   6. The receptor targeting conjugate of any one of clauses 1-5,    wherein each L², L⁵, and L⁸ is independently —C(═O)NR¹— or    —NR¹C(═O)—.

-   7. The receptor targeting conjugate of clause 6, wherein each L²,    L⁵, and L⁸ is —C(═O)NH—.

-   8. The receptor targeting conjugate of any one of clauses 1-7,    wherein each L³, L⁶, and L⁹ is independently substituted or    unsubstituted C₁-C₁₂ alkylene.

-   9. The receptor targeting conjugate of clause 8, wherein each L³ is    substituted or unsubstituted C₂-C₆ alkylene.

-   10. The receptor targeting conjugate of clause 8, wherein L³ is C₄    alkylene.

-   11. The receptor targeting conjugate of any one of clauses 1-10,    wherein each L⁶ and L⁹ is independently substituted or unsubstituted    C₂-C₁₀ alkylene.

-   12. The receptor targeting conjugate of clause 11, wherein each L⁶    and L⁹ is independently substituted or unsubstituted C₂-C₆ alkylene.

-   13. The receptor targeting conjugate of clause 11, wherein each L⁶    and L⁹ is C₃ alkylene.

-   14. A receptor targeting conjugate, comprising a compound of Formula    (VI):

-   -   wherein,    -   A is a receptor targeting moiety;    -   each L¹, L², L³, L⁴, L⁵, L⁶, L⁷, L⁸, L⁹, L¹⁰, and L¹¹, is        independently substituted or unsubstituted C₁-C₁₂ alkylene,        substituted or unsubstituted C₁-C₁₂ heteroalkylene, substituted        or unsubstituted C₂-C₁₂ alkenylene, substituted or unsubstituted        C₂-C₁₂ alkynylene, —(CH₂CH₂O)_(m)—, —(OCH₂CH₂)_(m)—, —O—, —S—,        —S(═O)—, —S(═O)₂—, —S(═O)(═NR¹)—, —C(═O)—, —C(═N—OR¹)—,        —C(═O)O—, —OC(═O)—, —C(═O)C(═O)—, —C(═O)NR¹—, —NR¹C(═O)—,        —OC(═O)NR¹—, —NR¹C(═O)O—, —NR¹C(═O)NR¹—, —C(═O)NR¹C(═O)—,        —S(═O)₂NR¹—, —NR¹S(═O)₂—, —NR¹—, or —N(OR¹)—;    -   L¹¹ is —(CH₂CH₂O)_(n)— or —(OCH₂CH₂)_(n)—;    -   each R¹ is independently H or substituted or unsubstituted        C₁-C₆alkyl;    -   R is a lipophilic organic residue;    -   m is an integer selected from 1 to 10; and    -   n is an integer selected from 1 to 200.

-   15. The receptor targeting conjugate of clause 14, wherein each L¹,    L⁴, and L⁷ is independently substituted or unsubstituted C₁-C₁₂    alkylene or substituted or unsubstituted C₁-C₁₂ heteroalkylene.

-   16. The receptor targeting conjugate of clause 14 or 15, wherein    each L¹, L⁴, and L⁷ is independently substituted or unsubstituted    C₁-C₁₂ heteroalkylene.

-   17. The receptor targeting conjugate of clause 16, wherein each L¹,    L⁴, and L⁷ is independently substituted or unsubstituted C₁-C₁₂    heteroalkylene comprising 1-10 O atoms.

-   18. The receptor targeting conjugate of clause 17, wherein each L¹,    L⁴, and L⁷ is independently —(CH₂CH₂O)_(p1)—(CH₂)_(q1)—; wherein p1    is 1-8; and q1 is 1-6.

-   19. The receptor targeting conjugate of clause 18, wherein each L¹,    L⁴, and L⁷ is —(CH₂CH₂O)₃—(CH₂)₂—.

-   20. The receptor targeting conjugate of clause 14 or 15, wherein    each L¹, L⁴, and L⁷ is independently substituted or unsubstituted    C₁-C₁₂ alkylene.

-   21. The receptor targeting conjugate of clause 20, wherein each L¹,    L⁴, and L⁷ is independently substituted or unsubstituted C₂-C₆    alkylene.

-   22. The receptor targeting conjugate of clause 21, wherein each L¹,    L⁴, and L⁷ is C₄ alkylene.

-   23. The receptor targeting conjugate of any one of clauses 14-22,    wherein each L², L⁵, and L⁸ is independently —C(═O)NR¹—, —NR¹C(═O)—,    —OC(═O)NR¹—, —NR¹C(═O)O—, —NR¹C(═O)NR¹—, or —C(═O)NR¹C(═O)—.

-   24. The receptor targeting conjugate of clause 23, wherein each L²,    L⁵, and L⁸ is independently —C(═O)NR¹— or —NR¹C(═O)—.

-   25. The receptor targeting conjugate of clause 24, wherein each L²,    L⁵, and L⁸ is —NHC(═O)—.

-   26. The receptor targeting conjugate of clause 24, wherein each L²,    L⁵, and L⁸ is —C(═O)NH—.

-   27. The receptor targeting conjugate of any one of clauses 14-26,    wherein each L³, L⁶, and L⁹ is independently substituted or    unsubstituted C₁-C₁₂ heteroalkylene.

-   28. The receptor targeting conjugate of clause 27, wherein each L³,    L⁶, and L⁹ is independently substituted or unsubstituted C₁-C₁₂    heteroalkylene comprising 1-10 O atoms.

-   29. The receptor targeting conjugate of clause 27 or 28, wherein    each L³, L⁶, and L⁹ is independently    —(CH₂CH₂O)_(p2)—(CH₂CH₂CH₂O)_(q2)—; wherein p2 is 1-8; and q2 is    1-6.

-   30. The receptor targeting conjugate of clause 29, wherein each L³,    L⁶, and L⁹ is —(CH₂CH₂O)—(CH₂CH₂CH₂O)—.

-   31. The receptor targeting conjugate of any one of clauses 14-26,    wherein each L³, L⁶, and L⁹ is independently —(CH₂CH₂CH₂O)_(q3)—;    wherein q3 is 1-8.

-   32. The receptor targeting conjugate of clause 31, wherein each L³,    L⁶, and L⁹ is —(CH₂CH₂CH₂O)₂—.

-   33. The receptor targeting conjugate of any one of clauses 1-32,    wherein L¹⁰ is substituted or unsubstituted C₁-C₁₂ alkylene.

-   34. The receptor targeting conjugate of clause 33, wherein L¹⁰ is    substituted or unsubstituted C₁-C₄ alkylene.

-   35. The receptor targeting conjugate of clause 34, wherein L¹⁰ is C₂    alkylene.

-   36. The receptor targeting conjugate of any one of clauses 1-35,    wherein L¹¹ is —(OCH₂CH₂)_(n)—.

-   37. The receptor targeting conjugate of clause 36, wherein n is    1-100.

-   38. The receptor targeting conjugate of clause 37, wherein n is    2-50.

-   39. The receptor targeting conjugate of clause 38, wherein n is 2,    12, 37, or 45.

-   40. The receptor targeting conjugate of any one of clauses 1-39,    wherein L¹² is —O—, —C(═O)O—, —C(═O)NR¹—, —NR¹C(═O)—, or    —NR¹C(═O)O—.

-   41. The receptor targeting conjugate of clause 40, wherein L¹² is    —C(═O)O— or —NR¹C(═O)O—.

-   42. The receptor targeting conjugate of clause 40, wherein L¹² is    —C(═O)O—.

-   43. The receptor targeting conjugate of clause 40, wherein L¹² is    —NHC(═O)O—.

-   44. The receptor targeting conjugate of clause 40, wherein L¹² is    —NHC(═O)—.

-   45. The receptor targeting conjugate of any one of clauses 1-44,    wherein A binds to a lectin.

-   46. The receptor targeting conjugate of clause 45, wherein the    lectin is an asialoglycoprotein receptor (ASGPR).

-   47. The receptor targeting conjugate of any one of clauses 1-44,    wherein A is N-acetylgalactosamine (GalNAc) or a derivative thereof.

-   48. The receptor targeting conjugate of any one of clauses 1-44,    wherein A is

-   49. The receptor targeting conjugate of any one of clauses 1-48,    wherein each R¹ is independently H or —CH₃.-   50. The receptor targeting conjugate of any one of clauses 1-49,    wherein each R¹ is H.-   51. The receptor targeting conjugate of any one of clauses 1-50,    wherein the lipophilic organic residue comprises one or more of    fatty alcohols, fatty acids, glycerolipids, glycerophospholipids,    sphingolipids, saccharolipids, polyketides, sterol lipids, and    prenol lipids.-   52. The receptor targeting conjugate of any one of clauses 1-51,    wherein the lipophilic organic residue comprises one or more fatty    alcohols.-   53. The receptor targeting conjugate of clause 52, wherein each    fatty alcohol is independently a saturated, monounsaturated, or    polyunsaturated fatty alcohol.-   54. The receptor targeting conjugate of clause 52 or 53, wherein the    fatty alcohol comprises one or more a C₂-C₂₆ fatty alcohol.-   55. The receptor targeting conjugate of any one of clauses 52-54,    wherein the fatty alcohol comprises two or more a C₂-C₂₆ fatty    alcohol.-   56. The receptor targeting conjugate of any one of clauses 52-54,    wherein each fatty alcohol is a C12, C14, C16, C18, C20, or C22    fatty alcohol.-   57. The receptor targeting conjugate of any one of clauses 52-54,    wherein each fatty alcohol is independently docosahexaenol,    eicosapentaenol, oleyl alcohol, stearyl alcohol,    (9Z,12Z)-octadeca-9,12-dien-1-yl alcohol, (Z)-docos-13-en-1-yl    alcohol, docosanyl alcohol, (E)-octadec-9-en-1-yl alcohol, icosanyl    alcohol, (9Z,12Z,15Z)-octadeca-9,12,15-trien-1-yl alcohol, or    palmityl alcohol.-   58. The receptor targeting conjugate of any one of clauses 52-54,    wherein each fatty alcohol is a stearyl alcohol.-   59. The receptor targeting conjugate of any one of clauses 1-50,    wherein the lipophilic organic residue comprises one or more sterol    lipids.-   60. The receptor targeting conjugate of any one of clauses 1-49,    wherein the lipophilic organic residue comprises one or more of    vitamins.-   61. The receptor targeting conjugate of any one of clauses 1-49 or    60, wherein each vitamin is independently a vitamin A, vitamin D,    vitamin E, or vitamin K.-   62. A receptor targeting conjugate, comprising a compound from Table    4.-   63. A nanoparticle composition comprising:    -   a. one or more nucleic acid molecular entities; and    -   b. a receptor targeting conjugate of any one of the preceding        clauses.-   64. The nanoparticle composition of clause 63, wherein the receptor    targeting conjugate comprises from about 0.001 mol % to about 20 mol    % of the total lipid content present in the nanoparticle    composition.-   65. The nanoparticle composition of clause 63, wherein the receptor    targeting conjugate comprises from about 0.01 mol % to about 1 mol %    of the total lipid content present in the nanoparticle composition.-   66. The nanoparticle composition of any one of clauses 63 to 65,    further comprising a sterol or a derivative thereof, comprising from    10 mol % to 70 mol % of the total lipid content present in the    nanoparticle composition.-   67. The nanoparticle composition of clause 66, wherein the sterol or    the derivative thereof is cholesterol or a cholesterol derivative.-   68. The nanoparticle composition of clause 67, wherein the    cholesterol or the cholesterol derivative comprises from 20 mol % to    50 mol % of the total lipid content present in the nanoparticle    composition.-   69. The nanoparticle composition of any one of clauses 63 to 68,    further comprising a phospholipid, comprising from 1 mol % to 20 mol    % of the total lipid content present in the nanoparticle    composition.-   70. The nanoparticle composition of clause 69, wherein the    phospholipid comprises from about 5 mol % to about 15 mol % of the    total lipid content present in said nanoparticle composition.-   71. The nanoparticle composition of clause 69 or 70, wherein the    phospholipid is selected from    1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),    1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),    1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC),    2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC),    1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and    sphingomyelin.-   72. The nanoparticle composition of clause 69 or 70, wherein the    phospholipid is DSPC.-   73. The nanoparticle composition of any one of clauses 63 to 72,    further comprising a stealth lipid, comprising from 0.1 mol % to 6    mol % of the total lipid content present in the nanoparticle    composition.-   74. The nanoparticle composition of clause 73, wherein the stealth    lipid comprises about 2.0 mol % to about 2.5 mol % of the total    lipid content present in said nanoparticle composition.-   75. The nanoparticle composition of clause 73 or 74, wherein the    stealth lipid is a PEG-lipid that has a number average molecular    weight of from about 200 Da to about 5000 Da.-   76. The nanoparticle composition of any one of clauses 63 to 75,    further comprising an amino lipid, comprising from about 10 mol % to    about 60 mol % of the total lipid content present in the    nanoparticle composition.-   77. The nanoparticle composition of any one of clauses 63 to 76,    wherein the nanoparticle composition comprises an antioxidant.-   78. The nanoparticle composition of clause 77, wherein the    antioxidant comprises ethylenediaminetetraacetic acid (EDTA).-   79. The nanoparticle composition of any one of clauses 63 to 78,    wherein the one or more nucleic acid molecular entities comprise a    single guide RNA (sgRNA) or guide RNA (gRNA) targeting a disease    causing gene of interest produced in the hepatocytes.-   80. The nanoparticle composition of any one of clauses 63 to 78,    wherein the one or more nucleic acid molecular entities comprise an    mRNA that encodes a Cas nuclease.-   81. The nanoparticle composition of any one of clauses 63 to 80,    wherein at least one of the one or more nucleic acid molecular    entities comprises a chemical modification.-   82. The nanoparticle composition of clause 81, wherein the chemical    modification is a 2′-F modification, a phosphorothioate    internucleotide linkage modification, acyclic nucleotides, LNA, HNA,    CeNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl,    2′-deoxy, 2′-fluoro, 2′-O—N-methylacetamido (2′-O-NMA), a    2′-O-dimethylaminoethoxyethyl (2′-0-DMAEOE), 2′-0-aminopropyl    (2′-O-AP), 4′-O-methyl, or a 2′-ara-F modification.-   83. The nanoparticle composition of clause 81, wherein the chemical    modification is a 2′-O-methyl modification.-   84. A pharmaceutical composition comprising the receptor targeting    conjugate of clauses 1 to 62 or a nanoparticle composition of any    one of clauses 63 to 83, and an excipient or carrier.-   85. The pharmaceutical composition of clause 84, wherein the    pharmaceutical composition comprises an mRNA encoding a gene editor    nuclease.-   86. The pharmaceutical composition of clause 84 or 85, wherein the    pharmaceutical composition comprises one or more guide RNA    molecules.-   87. The pharmaceutical composition of clause 84 or 85, wherein said    pharmaceutical composition comprises two or more guide RNA    molecules.-   88. The pharmaceutical composition of clause 87, wherein said two or    more guide RNA molecules target two or more genes of interest.-   89. The pharmaceutical composition of any one of clauses 84 to 88,    wherein the mRNA encodes Cas9 nuclease.-   90. The pharmaceutical composition of any one of clauses 84 to 88,    wherein the mRNA encodes a base editor nuclease.-   91. The pharmaceutical composition of any one of clauses 86 to 90,    wherein the mRNA and the one or more guide RNA molecules are present    in the same nanoparticle composition.-   92. The pharmaceutical composition of any one of clauses 86 to 90,    wherein the mRNA and the one or more guide RNA molecules are present    in different nanoparticle compositions.-   93. The pharmaceutical composition of any one of clauses 86 to 92,    wherein a ratio of the gRNA molecules to the mRNA in the    pharmaceutical composition is from about 0.01 to about 100 by weight    or by mole.-   94. The pharmaceutical composition of clause 93, wherein a ratio of    said gRNA molecules to said mRNA in said pharmaceutical composition    is about 50:1, about 40:1, about 30:1, about 20:1, about 18:1, about    16:1, about 14:1, about 12:1, about 10:1, about 9:1, about 8:1,    about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1,    about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6,    about 1:7, about 1:8, about 1:9, or about 1:10 by weight or by mole.-   95. A pharmaceutical composition comprising:    -   a. a first receptor targeting conjugate of any one of clauses        1-62 or a first nanoparticle composition of any one of clauses        63-83, and    -   b. a second receptor targeting conjugate of any one of clauses        1-62 or a second nanoparticle composition of any one of clauses        63-83.-   96. The pharmaceutical composition of clause 95, wherein said first    nanoparticle composition comprises a gene editor mRNA.-   97. The pharmaceutical composition of clause 95 or 96, wherein said    second nanoparticle composition comprises one or more guide RNA    molecules.-   98. The pharmaceutical composition of any one of clauses 95 to 97,    wherein a ratio of guide RNA molecules to mRNA in said    pharmaceutical composition is about 50:1, about 40:1, about 30:1,    about 20:1, about 18:1, about 16:1, about 14:1, about 12:1, about    10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about    4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about    1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about    1:10 by weight or by mole.-   99. A method of delivering a nucleic acid to a cell, the method    comprising contacting the cell with a nanoparticle composition of    any one of clauses 63-83 or a pharmaceutical composition of any one    of clauses 84-98, whereby the nucleic acid is delivered to said    cell.-   100. The method of clause 99, wherein said cell is contacted in    vivo, ex vivo, or in intro.-   101. A method of producing a polypeptide of interest in a cell, the    method comprising contacting said cell with a nanoparticle    composition of any one of clauses 63-83 or a pharmaceutical    composition of any one of clauses 84-98, whereby the nucleic acid is    capable of being translated in said cell to produce the polypeptide.-   102. A method of treating a disease or condition in a subject, the    method comprising administering to the subject a therapeutically    effective amount of a pharmaceutical composition of any one of    clauses 84-98.-   103. The method of clause 102, wherein the disease or condition is a    coronary disease.-   104. The method of clause 102 or 103, wherein the subject is    low-density lipoprotein receptor (LDLR)-deficient.-   105. A method of delivering a nucleic acid molecular entity to the    liver of a subject, comprising administering to the subject a    pharmaceutical composition of any one of clauses 84 to 98, thereby    delivering the nucleic acid molecular entity.-   106. A nucleotide conjugate comprising:    -   (a) a nucleic acid, and    -   (b) a targeting moiety connected to the nucleic acid in (a),        wherein the targeting moiety comprises a structure of Table 1.-   107. The nucleotide conjugate of clause 106, wherein the targeting    moiety further comprises a coupling sequence that hybridizes with    the nucleic acid in (a).-   108. A nucleotide conjugate comprising:    -   (a) a nucleic acid, and    -   (b) a targeting moiety connected to the nucleic acid in (a),        wherein the targeting moiety comprises a coupling sequence that        hybridizes with the nucleic acid in (a).-   109. The nucleotide conjugate of any one of clauses 106-108, wherein    the nucleic acid comprises a single stranded, double stranded, a    partially double stranded, or a hairpin stem-loop nucleic acid, and    wherein the targeting moiety is a receptor targeting moiety.-   110. The nucleotide conjugate of any one of clauses 106-109, wherein    the targeting moiety binds to a lectin.-   111. The nucleotide conjugate of clause 110, wherein the lectin is    an asialoglycoprotein receptor (ASGPR).-   112. The nucleotide conjugate of clause 111, wherein the targeting    moiety comprises one or more N-acetylgalactosamine (GalNAc) or    GalNAc derivatives.-   113. The nucleotide conjugate of any one of clauses 106-112, wherein    the targeting moiety comprises a spacer.-   114. The nucleotide conjugate of clause 113, wherein the spacer    comprises polyethylene glycol, substituted or unsubstituted C₁-C₁₂    alkylene, or both, wherein the polyethylene glycol has from 1 to 5    repeating units.-   115. The nucleotide conjugate of any one of clauses 106-114, wherein    the targeting moiety is linked to one or more strands of the nucleic    acid through one or more linkers.-   116. The nucleotide conjugate of any one of clauses 108-115, wherein    the targeting moiety comprises a structure of Table 1.-   117. The nucleotide conjugate of any one of clauses 107-116, wherein    the coupling sequence hybridizes with the nucleic acid in (a).-   118. The nucleotide conjugate of clause 117, wherein the coupling    sequence hybridizes with an extension in the nucleic acid in (a).-   119. The nucleotide conjugate of any one of clauses 106-118, wherein    the targeting moiety is attached to the 5′ end of the nucleic acid    sequence, the 3′ end of the nucleic acid sequence, or the middle of    the nucleic acid sequence.-   120. The nucleotide conjugate of any one of clauses 106-119, wherein    the targeting moiety comprises at least two GalNAcs or GalNAc    derivatives.-   121. The nucleotide conjugate of any one of clauses 106-120, wherein    the targeting moiety comprises at least three GalNAcs or GalNAc    derivatives.-   122. The nucleotide conjugate of any one of clauses 114-121, wherein    the GalNAcs or GalNAc derivatives are connected to the nucleic acid    in (a) via a linker in the targeting moiety, via hybridization of    the coupling sequence in the targeting moiety that hybridizes with    the nucleic acid in (a), or via a combination thereof.-   123. The nucleotide conjugate of any one of clauses 107-122, wherein    the targeting moiety comprises at least two coupling sequences that    hybridize with the nucleic acid in (a).-   124. The nucleotide conjugate of clause 123, wherein the at least    two coupling sequences are identical.-   125. The nucleotide conjugate of clause 123, wherein the at least    two coupling sequences are different.-   126. The nucleotide conjugate of any one of clauses 106-125, further    comprising a second targeting moiety.-   127. The nucleotide conjugate of clause 126, wherein the second    targeting moiety binds to an asialoglycoprotein receptor (ASGPR).-   128. The nucleotide conjugate of clause 127, wherein the second    targeting moiety is linked to one or more strands the nucleic acid    through a spacer and/or through one or more linkers.-   129. The nucleotide conjugate of clause 128, wherein the second    targeting moiety comprises a GalNAc or GalNAc derivative.-   130. The nucleotide conjugate of clause 129, wherein the second    targeting moiety comprises at least three GalNAcs or GalNAc    derivatives.-   131. The nucleotide conjugate of clause 129 or 130, wherein the    GalNAc or GalNAc derivatives are connected to the nucleic acid    in (a) via a linker in the targeting moiety, via hybridization of    the coupling sequence in the targeting moiety that hybridizes with    the nucleic acid in (a), or via a combination thereof.-   132. The nucleotide conjugate of any one of clauses 126-131, wherein    the second targeting moiety comprises a structure of Table 1.-   133. The nucleotide conjugate of any one of clauses 126-132, wherein    the second targeting moiety comprises a coupling sequence that    hybridizes with the nucleic acid.-   134. The nucleotide conjugate of any one of clauses 126-133, wherein    the second targeting moiety is attached to the 5′ end of the nucleic    acid, the 3′ end of the nucleic acid, or the middle of the nucleic    acid.-   135. The nucleotide conjugate of any one of clauses 106-134, wherein    the nucleic acid in (a) comprises RNA or DNA.-   136. The nucleotide conjugate of any one of clauses 107-135, wherein    the coupling sequence comprises RNA, DNA, chemically modified RNA,    chemically modified DNA, or a hybrid of DNA and RNA.-   137. The nucleotide conjugate of any one of clauses 107-136, wherein    the coupling sequence comprises one or more of (a), (c), (g), (u),    (A)n, (T)n, (U)n, (a)n, or (u)n, wherein n is an integer no less    than 3, wherein a is 2′-O-methyladenosine (2′-OMe A), wherein c is    2′-O-methylacytidine (2′-OMe-C), wherein g is 2′-O-methylacytidine    guanine (2′-OMe-G), and wherein u is 2′-O-methyluridine (2′-OMe-U).-   138. The nucleotide conjugate of any one of clauses 107-137, wherein    the coupling sequence comprises one or more of (a), (c), (g), (u),    (A)n, (T)n, (U)n, (a)n, or (u)n, wherein n is an integer no less    than 3, wherein a is 2′-O-methyladenosine (2′-OMe A), wherein c is    2′-O-methylacytidine (2′-OMe-C), wherein g is 2′-O-methylacytidine    guanine (2′-OMe-G), and wherein u is 2′-O-methyluridine (2′-OMe-U).-   139. The nucleotide conjugate of clause 137 or 138, wherein the (a),    (c), (g), or (u) is scattered along the nucleic acid or the coupling    sequence.-   140. The nucleotide conjugate of any one of clauses 137-139, wherein    the nucleic acid and the coupling sequence comprise one or more G-C    base pairing within a hybridization duplex wherein the coupling    sequence hybridizes with the nucleic acid and wherein said one or    more G-C base pairing increases stability of the hybridization    duplex.-   141. The nucleotide conjugate of any one of clauses 115-140, wherein    the linker comprises a covalent linker.-   142. The nucleotide conjugate of any one of clauses 115-140, wherein    the linker comprises a non-covalent linker.-   143. The nucleotide conjugate of any one of clauses 115-142, wherein    the linker comprises a monovalent linker, a bivalent linker, a    trivalent linker, or a combination thereof.-   144. The nucleotide conjugate of any one of clauses 115-143, wherein    the linker comprises a biocleavable linker.-   145. The nucleotide conjugate of any one of clauses 115-143, wherein    the linker comprises a non-biocleavable linker.-   146. The nucleotide conjugate of clause 142, wherein the linker    comprises a phosphate, phosphorothioate, amide, ether, oxime,    hydrazine or carbamate.-   147. The nucleotide conjugate of clause 146, wherein the linker is a    phosphate or phosphorothioate.-   148. The nucleotide conjugate of any one of clauses 106-147, wherein    the nucleic acid in (a) comprises a chemical modification.-   149. The nucleotide conjugate of clause 148, wherein the nucleic    acid in (a) comprises a 2′-F modification, a phosphorothioate    internucleotide linkage modification, acyclic nucleotides, LNA, HNA,    CeNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl,    2′-deoxy, 2′-fluoro, 2′-O—N-methylacetamido (2′-O-NMA), a    2′-O-dimethylaminoethoxyethyl (2′-0-DMAEOE), 2′-0-aminopropyl    (2′-O-AP), 4′-O-methyl, or a 2′-ara-F modification.-   150. The nucleotide conjugate of clause 149, wherein the nucleic    acid comprises a 2′-O-methyl modification.-   151. The nucleotide conjugate of clause 149, wherein the nucleic    acid comprises a phosphorothioate internucleotide linkage    modification.-   152. The nucleotide conjugate of any one of clauses 106-151, wherein    the nucleic acid is capable of hybridizing with a target sequence    within a target gene of a genome.-   153. The nucleotide conjugate of clause 152, wherein the nucleic    acid comprises a mRNA, siRNA, shRNA, antisense oligonucleotide,    microRNA, anti-microRNA or antimir, supermir, antagomir, ribozyme,    triplex-forming oligonucleotide, decoy oligonucleotide,    splice-switching oligonucleotide, immunostimulatory oligonucleotide,    RNA activator, Ul adaptor, guide RNA, or any combinations thereof-   154. The nucleotide conjugate of clause 153, wherein the nucleic    acid encodes a protein.-   155. The nucleotide conjugate of clause 154, wherein the nucleic    acid is a CRISPR enzyme.-   156. The nucleotide conjugate of clause 153, wherein the nucleic    acid is a guide RNA capable of forming a complex with a CRISPR    enzyme.-   157. The nucleotide conjugate of clause 156 wherein the guide RNA is    a single guide RNA or a dual guide RNA.-   158. The nucleotide conjugate of clause 157, wherein the CRISPR    enzyme is selected from the group consisting of Cas9, Cpf1, CasX,    CasY, C2c1, C2c3, and base editor fusion protein.-   159. The nucleotide conjugate of any one of clauses 158, wherein the    nucleic acid further comprises a mRNA encoding the CRISPR enzyme.-   160. The nucleotide conjugate of clause 159, wherein the CRISPR    enzyme results in an alteration in the target sequence.-   161. The nucleotide conjugate of any one of clauses 152-160, wherein    the target gene is involved in a lipid metabolism pathway.-   162. the nucleotide conjugate of clause 161, wherein the target gene    is selected from the group consisting of PCSK9, ANGPTL3, APOC3, LPA,    APOB, MTP, ANGPTL4, ANGPTL8, APOA5, APOE, LDLR, IDOL, NPC1L1, ASGR1,    TM6SF2, GALNT2, GCKR, LPL, MLXIPL, SORT1, TRIB1, MARC1, ABCG5, and    ABCG8-   163. A particle comprising the nucleotide conjugate and the CRISPR    enzyme of any one of clauses 155-162.-   164. The particle of clause 163, wherein the particle is a lipid    nanoparticle, a liposome, an inorganic nanoparticle, or an RNP.-   165. A cell comprising the nucleotide conjugate of any one of    clauses 106-162.-   166. The cell of clause 165, wherein the cell is a prokaryotic cell,    a eukaryotic cell, a vertebrate cell, a mouse cell, a non-human    primate cell, or a human cell.-   167. A pharmaceutical composition comprising the nucleotide    conjugate of any one of clauses 106-162, the particle of clause 163    or 164, or the cell of clause 165 or 166.-   168. The pharmaceutical composition of clause 167, further    comprising a pharmaceutically acceptable adjuvant, diluent, carrier,    preservative, excipient, buffer, stabilizer, or a combination    thereof.-   169. The pharmaceutical composition of clause 168, wherein the    carrier comprises solvents, dispersion media, dispersion or    suspension aids, surface active agents, isotonic agents, thickening    or emulsifying agents, preservatives, lipids, lipidoids, polymers,    lipoplexes, core-shell nanoparticles, hyaluronidase, nanoparticle    mimics, or combinations thereof.-   170. A kit comprising the nucleotide conjugate of any one of clauses    106-162.-   171. A method for reducing the risk of coronary disease in a subject    in need thereof, comprising administering to the subject an    effective amount of the nucleotide conjugate of any one of clauses    106-162.-   172. A method for reducing the risk of coronary disease in a subject    in need thereof, comprising administering to the subject an    effective amount of a pharmaceutical composition, said    pharmaceutical composition comprising    -   (a) a nucleic acid, and    -   (b) a targeting moiety connected to the nucleic acid in (a),        wherein the targeting moiety comprises a structure of Table 1.-   173. A method of delivering a nucleic acid to the liver of a    subject, comprising administering to the subject said nucleic acid    connected to a targeting moiety, wherein the targeting moiety    comprises a structure of Table 1.-   174. The method of clause 172 or 173, wherein the targeting moiety    further comprises a coupling sequence that hybridizes with the    nucleic acid in (a).-   175. A method for reducing the risk of coronary disease in a subject    in need thereof, comprising administering to the subject an    effective amount of a pharmaceutical composition, said    pharmaceutical composition comprising    -   (a) a nucleic acid, and    -   (b) a targeting moiety connected to the nucleic acid in (a),        wherein the targeting moiety comprises a coupling sequence that        hybridizes with the nucleic acid in (a).-   176. A method of delivering a nucleic acid to the liver of a    subject, comprising administering to the subject said nucleic acid    connected to a targeting moiety, wherein the targeting moiety    comprises a coupling sequence that hybridizes with the nucleic acid.-   177. The method of any one of the clauses 172-176, wherein the    nucleic acid comprises a single stranded, double stranded, a    partially double stranded, or a hairpin stem-loop nucleic acid, and    wherein the targeting moiety is a receptor targeting moiety.-   178. The method of any one of clauses 172-177, wherein the targeting    moiety binds to a lectin.-   179. The method of clause 178, wherein the lectin is an    asialoglycoprotein receptor (ASGPR).-   180. The method of any one of clauses 172-179, wherein the targeting    moiety comprises one or more N-acetylgalactosamine (GalNAc) or    GalNAc derivatives.-   181. The method of clause 180, wherein the targeting moiety    comprises at least three GalNAc or GalNAc derivatives.-   182. The method of any one of clauses 172-181, wherein the nucleic    acid comprises (i) a guide RNA and a nuclease mRNA or (ii) a guide    RNA complexed in a nuclease RNP, and wherein the guide RNA is    capable of directing the nuclease to a target sequence in a target    gene.-   183. The method of clause 182, wherein the guide RNA comprises a    single guide RNA or a dual guide RNA.-   184. The method of clause 183, wherein the nuclease is a CRISPR    enzyme.-   185. The method of clause 184, wherein the CRISPR enzyme selected    from the group consisting of Cas9, Cpf1, CasX, CasY, C2c1, C2c3, and    base editor fusion protein.-   186. The method of clause 185, wherein the CRISPR enzyme results in    an alteration in the target sequence.-   187. The method of any one of clauses 182-186, wherein the    administration results in reduced expression of the target gene in    the liver of the subject.-   188. The method of clause 186 or 187, wherein expression of the    target gene in the liver of the subject is reduced by at least 1%,    at least 5%, at least 10%, at least 20%, at least 30%, at least 40%,    at least 50%, at least 60%, at least 70%, at least 80%, at least    85%, at least 90%, at least 95%, at least 96%, at least 97%, at    least 98%, at least 99%, or greater than 99.99% as compared to a    control tissue of the subject.-   189. The method of any one of clauses 182-188, wherein the target    gene is associated with a coronary disease.-   190. The method of clause 189, wherein the target gene is selected    from the group consisting of PCSK9, ANGPTL3, APOC3, LPA, APOB, MTP,    ANGPTL4, ANGPTL8, APOA5, APOE, LDLR, IDOL, NPC1L1, ASGR1, TM6SF2,    GALNT2, GCKR, LPL, MLXIPL, SORT1, TRIB1, MARC1, ABCG5, and ABCG8.-   191. The method of any one of clauses 182-190, wherein the guide RNA    comprises a sequence selected from the group consisting of SEQ ID    NOs 1-23.-   192. The method of any one of clauses 174-191, wherein the coupling    sequence comprises RNA, DNA, chemically modified RNA, chemically    modified DNA, or a hybrid of DNA and RNA.-   193. The method of any one of clauses 174-192, wherein the coupling    sequence comprises (A)n, (T)n, (U)n, (a)n, or (u)n, wherein n is an    integer no less than 3, wherein a is 2′-O-methyladenosine (2′-OMe    A), and wherein u is 2′-O-methyluridine (2′-OMe-U).-   194. The method of any one of clauses 172-193, wherein the nucleic    acid in (a) comprises (A)n, (T)n, (U)n, (a)n, or (u)n, wherein n is    an integer no less than 3, wherein a is 2′-O-methyladenosine, and    wherein u is 2′-O-methyluridine.-   195. The method of any one of clauses 172-194, wherein the targeting    moiety is linked to the nucleic acid in (a) via a linker in the    targeting moiety, via hybridization of the coupling sequence in the    targeting moiety that hybridizes with the nucleic acid in (a), or    via a combination thereof.-   196. The method of clauses 195, wherein the linker comprises a    covalent linker.-   197. The method of clause 196, wherein the linker comprises a    phosphate, phosphorothioate, amide, ether, oxime, hydrazine or    carbamate.-   198. The method of clause 197, wherein the linker is a phosphate or    phosphorothioate.-   199. The method of any one of 172-198, wherein the nucleic acid    in (a) comprises a chemical modification.-   200. The method of clause 199, wherein the nucleic acid in (a)    comprises a 2′-F modification, a phosphorothioate internucleotide    linkage modification, acyclic nucleotides, LNA, HNA, CeNA,    2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy,    2′-fluoro, 2′-O—N-methylacetamido (2′-O-NMA), a    2′-O-dimethylaminoethoxyethyl (2′-0-DMAEOE), 2′-0-aminopropyl    (2′-O-AP), or a 2′-ara-F modification.-   201. The method of clause 200, wherein the nucleic acid comprises a    2′-O-methyl modification.-   202. The method of clause 201, wherein the nucleic acid comprises a    phosphorothioate internucleotide linkage modification.-   203. The method of any one of clauses 172-202, where in the level of    the nucleic acid in the liver of the subject is at least 1.5, at    least 2, at least 2.5, at least 3, at least 5, at least 10, at least    15, at least 20, at least 30, at least 40, at least 50 folds higher    as compared to other tissues of the subject at least 1 hours, 2    hours, 6 hours, 12 hours, 24 hours, 2 days, 1 week, 2 weeks, 3    weeks, 6 weeks, or 8 weeks post delivery.-   204. The method of clause any one of clauses 175-203, wherein the    effective amount is about 1 mg/kg to about 10 mg/kg.-   205. The method of clause 175-204, wherein the administration    results in reduced blood triglycerides and/or reduced low-density    lipo-protein cholesterol in the subject in need thereof.-   206. The method of any one of clauses 175-205, wherein the    administration is performed intravenously, intrathecally,    intramuscularly, intraventricularly, intracerebrally,    intracerebellarly, intracerebroventricularly, intraperenchymally,    subcutaneously, or a combination thereof.-   207. A method for reducing the risk of coronary disease in a subject    in need thereof, the method comprising administering to the subject    a pharmaceutical composition comprising    -   (a) (i) a single guide RNA and a nuclease mRNA, (ii) a dual        guide RNA and a nuclease mRNA, (iii) a single guide RNA and an        RNP, or (iv) a dual guide RNAs and an RNP; and    -   (b) a asialoglycoprotein receptor (ASGPR) targeting moiety        connected to the nucleic acid in (a),    -   wherein the single guide RNA or the dual guide RNA comprises 4        or more 2′-O-methyl modifications and 2 or more phosphorothioate        internucleotide linkages, wherein the targeting moiety comprises        a structure of Table 1, and wherein the guide RNA hybridizes        with a PCSK9 gene.-   208. A method for reducing the risk of coronary disease in a subject    in need thereof, the method comprising administering to the subject    a pharmaceutical composition comprising    -   (a) (i) a single guide RNA and a nuclease mRNA, (ii) a dual        guide RNA and a nuclease mRNA, (iii) a single guide RNA and an        RNP, or (iv) a dual guide RNAs and an RNP; and    -   (b) a targeting moiety connected to the nucleic acid in (a),    -   wherein the single guide RNA or the dual guide RNA comprises 4        or more 2′-O-methyl modifications and two or more        phosphorothioate internucleotide linkages, wherein the targeting        moiety comprises a coupling sequence that hybridizes with the        single guide RNA in (a), and wherein the guide RNA hybridizes        with a PCSK9 gene.-   209. The method of clause 207 or 208, wherein the nuclease mRNA    and/or the single guide RNA comprises at least one chemical    modification.-   210. The method of clause 209, wherein the chemical modification is    selected from the group consisting of a 2′-F modification,    phosphorothioate internucleotide linkage modification, acyclic    nucleotides, LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-methyl,    2′-O-allyl, 2′-C-allyl, 2′-deoxy, 2′-fluoro, 2′-O—N-methylacetamido    (2′-O-NMA), a 2′-O-dimethylaminoethoxyethyl (2′-0-DMAEOE),    2′-0-aminopropyl (2′-O-AP), 4′-O-methyl, and a 2′-ara-F    modification.-   211. The method of clause 210, wherein administrating of the nucleic    acid conjugate results in a reduced level of immune response as    compared to a control nucleic acid conjugate without said chemical    modification.-   212. A nucleotide conjugate comprising a structure of Formula (IV)

-   -   wherein each X is independently H or a protecting group, R^(A)        is —OX or —NHAc, Y is O or S, and W represents    -   (a) (i) a single guide RNA and a nuclease mRNA, (ii) a dual        guide RNA and a nuclease mRNA, (iii) a single guide RNA and an        RNP, or (iv) a dual guide RNAs and an RNP; or    -   (b) a coupling sequence.

-   213. The nucleotide conjugate of clause 212, wherein the one or more    linkers comprise a structure selected from the group consisting of:

-   214. The nucleotide conjugate of clause 212 or 213, wherein each of    the linkers independently has a structure of    -(L¹)_(k1)-(L²)_(k2)-(L³)_(k3)-(L⁴)_(k4)-, wherein each of k1, k2,    k3, and k4 is independently 0, 1 or 2, and each of the L¹, L², L³    and L⁴ is independently selected from —O—, —S—, S(═O)₁₋₂—, —C(═O)—,    —C(═S)—, —NR^(L)—, —OC(═O)—, —C(═O)O—, —OC(═O)O—, —C(═O)NR^(L)—,    —OC(═O)NR^(L)—, —NR^(L)C(═O)—, —NR^(L)C(═O) NR^(L)—, —P(═O)R^(L)—,    —NR^(L)S(═O)(═NR^(L))—, —NR^(L)S(═O)₂—, —S(═O)₂NR^(L)—, —N═N—,    —(CH₂—CH₂—O)₁₋₆—, linear or branched C₁₋₆ alkylene, linear or    branched C₂₋₆ alkenylene, linear or branched C₂₋₆ alkynylene, C₃-C₈    cycloalkylene, C₂-C₇ heterocycloalkylene, C₆-C₁₀ arylene, and C₅-C₉    heteroarylene, wherein the alkylene, alkenylene, alkynylene,    cycloalkylene, cycloalkylene, arylene, or heteroarylene is    substituted or unsubstituted, and wherein each R^(L) is    independently H, D, cyano, halogen, substituted or unsubstituted    C₁-C₆ alkyl, —CD₃, —OCH₃, —OCD₃, substituted or unsubstituted C₁-C₆    haloalkyl, substituted or unsubstituted C₁-C₆ heteroalkyl,    substituted or unsubstituted C₃-C₈ cycloalkyl, substituted or    unsubstituted C₂-C₇ heterocycloalkyl, substituted or unsubstituted    aryl, or substituted or unsubstituted heteroaryl. In some    embodiments, each R^(L) is independently H, substituted or    unsubstituted C₁-C₆ alkyl, —OCH₃, substituted or unsubstituted C₁-C₆    haloalkyl, substituted or unsubstituted C₁-C₆ heteroalkyl,    substituted or unsubstituted C₃-C₈cycloalkyl, or substituted or    unsubstituted C₂-C₇ heterocycloalkyl.-   215. The nucleotide conjugate of any one of clauses 212 to 214,    wherein the sum of k1, k2, k3, and k4 is 1, 2, or 3.-   216. A method of preparing a formulation comprising nanoparticles,    wherein the nanoparticles comprise (i) one or more nucleic acid    molecular entities, (ii) one or more lipids selected from a sterol    or a derivative thereof, a phospholipid, a stealth lipid, and an    amino lipid, and (iii) a receptor targeting conjugate, the method    comprising:    -   a. providing a first solution comprising the one or more nucleic        acid molecular entities;    -   b. providing a second solution comprising at least one of the        one or more lipids;    -   c. mixing the first solution and the second solution, thereby        producing a mixture comprising nanoparticles that comprise the        one or more nucleic acid molecular entities and the one or more        lipids;    -   d. mixing the receptor targeting conjugate with the        nanoparticles produced in step (c);    -   e. incubating the nanoparticles; and    -   f. optionally carrying out a buffer exchange process.-   217. The method of clause 216, wherein the receptor targeting    conjugate is combined with the one or more lipids after the mixing    step in (c).-   218. The method of clause 216, wherein the receptor targeting    conjugate is added in a dilution buffer, and wherein the dilution    buffer is mixed with preformed nucleic acid-lipid nanoparticles    coming out of an inline mixing chamber thereby forming the    nanoparticles.-   219. The method of clause 216, wherein the receptor targeting    conjugate is introduced after an addition of a dilution buffer to    the mixture and holding the diluted mixture for a period of time.-   220. The method of clause 219, wherein the holding time is between 1    and 120 minutes.-   221. The method of clause 219, wherein the holding time is between 1    and 90 minutes, between 1 and 60 minutes, or between 10 and 40    minutes.-   222. The method of clause 219, wherein the holding time is about 30    minutes.-   223. The method of clause 216, wherein the receptor targeting    conjugate is introduced to the nanoparticles after buffer exchange.-   224. The method of clause 216, wherein the receptor targeting    conjugate is introduced to the nanoparticles after buffer exchange    and concentration, but prior to storage.-   225. The method of clause 216, wherein the receptor targeting    conjugate is introduced to the nanoparticles after storage and    thawing, and prior to dosing or evaluation.-   226. A method of preparing a formulation comprising nanoparticles,    wherein the nanoparticles comprise (i) one or more nucleic acid    molecular entities, (ii) one or more lipids selected from a sterol    or a derivative thereof, a phospholipid, a stealth lipid, and an    amino lipid, and (iii) a receptor targeting conjugate, the method    comprising:    -   a. providing a first solution comprising the one or more nucleic        acid molecular entities;    -   b. providing a second solution comprising at least one of the        one or more lipids;    -   c. inline mixing of the first solution and the second solution,        thereby producing a mixture comprising nanoparticles that        comprise the one or more nucleic acid molecular entities and the        one or more lipids;    -   d. inline mixing of the receptor targeting conjugate to the        mixture of step (c), thereby producing a mixture comprising        nanoparticles that comprise the one or more nucleic acid        molecular entities, the one or more lipids, and the receptor        targeting conjugate;    -   e. diluting the mixture of step (d) by adding a dilution buffer;        and    -   f. optionally carrying out a buffer exchange process.-   227. The method of clause 226, wherein the inline mixing of step (c)    and the inline mixing of step (d) are performed successively.-   228. A method of preparing a formulation comprising nanoparticles,    wherein the nanoparticles comprise (i) one or more nucleic acid    molecular entities, (ii) one or more lipids selected from a sterol    or a derivative thereof, a phospholipid, a stealth lipid, and an    amino lipid, and (iii) a receptor targeting conjugate, the method    comprising:    -   a. providing a first solution comprising the one or more nucleic        acid molecular entities;    -   b. providing a second solution comprising (i) at least one of        the one or more lipids and (ii) at least a portion of the        receptor targeting conjugate;    -   c. mixing the first solution and the second solution, thereby        producing a mixture comprising nanoparticles;    -   d. optionally incubating the nanoparticles; and    -   e. optionally carrying out a buffer exchange process.-   229. The method of clause 228, wherein the second solution comprises    all the receptor targeting conjugate.-   230. A method of preparing a formulation comprising nanoparticles,    wherein the nanoparticles comprise (i) one or more nucleic acid    molecular entities, (ii) one or more lipids selected from a sterol    or a derivative thereof, a phospholipid, a stealth lipid, and an    amino lipid, and (iii) a receptor targeting conjugate, the method    comprising:    -   a. providing a first solution comprising the one or more nucleic        acid molecular entities;    -   b. providing a second solution comprising at least one of the        one or more lipids;    -   c. mixing the first solution and the second solution, thereby        producing a mixture comprising nanoparticles that comprise the        one or more nucleic acid molecular entities and the one or more        lipids;    -   d. combining the receptor targeting conjugate with the one or        more lipids, wherein at least a portion of the receptor        targeting conjugate is combined with the one or more lipids        prior to or concurrently with the mixing step;    -   e. optionally incubating the nanoparticles; and    -   f. optionally carrying out a buffer exchange process.-   231. The method of clause 230, wherein at least a portion of the    receptor targeting conjugate is combined with the one or more lipids    concurrently with the mixing step.-   232. The method of clause 230, wherein at least a portion of the    receptor targeting conjugate is combined with the one or more lipids    prior to the mixing step.-   233. The method of clause 232, wherein the receptor targeting    conjugate is combined with the one or more lipids in the second    solution.-   234. The method of clause 228 or 230, wherein a portion of the    receptor targeting conjugate is combined with the one or more lipids    in the second solution and a portion of the receptor targeting    conjugate is combined with the one or more lipids after the mixing.-   235. The method of clause 228 or 230, wherein a portion of the    receptor targeting conjugate is combined with the one or more lipids    in the second solution and a portion of the receptor targeting    conjugate is combined with the one or more lipids after the    incubating step.-   236. The method of clause 228 or 230, wherein a portion of the    receptor targeting conjugate is combined with the one or more lipids    in the second solution and a portion of the receptor targeting    conjugate is combined with the one or more lipids after the buffer    exchange step.-   237. The method of any one of clauses 228 or 230, further comprising    diluting the mixture produced by mixing the first and the second    solutions by adding a dilution buffer.-   238. The method of clause 237, wherein the mixture is diluted    inline.-   239. The method of clause 237 or 238, wherein the dilution buffer    comprises at least a portion of the receptor targeting conjugate.-   240. The method of any one of clauses 237 to 239, wherein the    dilution buffer comprises at least a portion of the stealth lipid.-   241. The method of any one of clauses 216 to 240, wherein the first    solution comprises an aqueous buffer.-   242. The method of any one of clauses 216 to 241, wherein the second    solution comprises ethanol.-   243. The method of any one of clauses 216 or 228 to 242, wherein the    mixing comprises laminar mixing, vortex mixing, turbulent mixing, or    a combination thereof-   244. The method of any one of clauses 216 or 228 to 242, wherein the    mixing comprises cross-mixing.-   245. The method of any one of clauses 216 or 228 to 242, wherein the    mixing comprises inline mixing.-   246. The method of any one of clauses 216 to 242, wherein the mixing    comprises introducing at least a portion of the first solution    through a first inlet channel and at least a portion of the second    solution through a second inlet channel, and wherein an angle    between the first inlet channel and the second inlet channel is from    about 15 to 180 degrees.-   247. The method of clause 246, wherein the mixing comprises    introducing a portion of the first solution through a third inlet    channel.-   248. The method of any one of clauses 216 to 247, wherein the buffer    exchange comprises dialysis, chromatography, or tangential flow    filtration (TFF).-   249. The method of any one of clauses 216 to 248, further comprising    a filtration step.-   250. The method of any one of clauses 216 to 249, wherein the    receptor targeting conjugate comprises one or more    N-acetylgalactosamine (GalNAc) or GalNAc derivatives.-   251. The method of clause 250, wherein the receptor targeting    conjugate is selected from Table 4.-   252. The method of any one of clauses 216 to 249, wherein the    receptor targeting conjugate is described in any one of clauses 1 to    62.-   253. The method of any one of clauses 216 to 249, wherein the    nanoparticles comprise a first nanoparticle composition of any one    of clauses 63-83-   254. The method of any one of clauses 216 to 249, wherein the    formulation is a pharmaceutical composition of any one of clauses 84    to 98.-   255. A pharmaceutical composition comprising nanoparticles, wherein    the nanoparticles comprise (i) one or more nucleic acid molecular    entities, (ii) one or more lipids selected from a sterol or a    derivative thereof, a phospholipid, a stealth lipid, and an amino    lipid, and (iii) a receptor targeting conjugate, wherein the    formulation is prepared by a method of any one of clauses 216 to    253.-   256. A pharmaceutical composition comprising nanoparticles, wherein    the nanoparticles comprise (i) one or more nucleic acid molecular    entities, (ii) one or more lipids selected from a sterol or a    derivative thereof, a phospholipid, a stealth lipid, and an amino    lipid, and (iii) a receptor targeting conjugate, wherein the    formulation is prepared by a method comprising:    -   a. providing a first solution comprising the one or more nucleic        acid molecular entities;    -   b. providing a second solution comprising at least one of the        one or more lipids;    -   c. mixing the first solution and the second solution, thereby        producing a mixture comprising nanoparticles that comprise the        one or more nucleic acid molecular entities and the one or more        lipids;    -   d. mixing the receptor targeting conjugate with the        nanoparticles produced in step (c);    -   e. incubating the nanoparticles; and    -   f. optionally carrying out a buffer exchange process.-   257. A pharmaceutical composition comprising nanoparticles, wherein    the nanoparticles comprise (i) one or more nucleic acid molecular    entities, (ii) one or more lipids selected from a sterol or a    derivative thereof, a phospholipid, a stealth lipid, and an amino    lipid, and (iii) a receptor targeting conjugate, wherein the    formulation is prepared by a method comprising:    -   a. providing a first solution comprising the one or more nucleic        acid molecular entities;    -   b. providing a second solution comprising at least one of the        one or more lipids;    -   c. mixing the first solution and the second solution, thereby        producing a mixture comprising nanoparticles that comprise the        one or more nucleic acid molecular entities and the one or more        lipids;    -   d. combining the receptor targeting conjugate with the one or        more lipids, wherein at least a portion of the receptor        targeting conjugate is combined with the one or more lipids        prior to or concurrently with the mixing step;    -   e. optionally incubating the nanoparticles; and    -   f. optionally carrying out a buffer exchange process.-   258. A pharmaceutical composition comprising nanoparticles, wherein    the nanoparticles comprise (i) one or more nucleic acid molecular    entities, (ii) one or more lipids selected from a sterol or a    derivative thereof, a phospholipid, a stealth lipid, and an amino    lipid, and (iii) a receptor targeting conjugate, wherein the    formulation is prepared by a method comprising:    -   a. providing a first solution comprising the one or more nucleic        acid molecular entities;    -   b. providing a second solution comprising at least one of the        one or more lipids;    -   c. inline mixing of the first solution and the second solution,        thereby producing a mixture comprising nanoparticles that        comprise the one or more nucleic acid molecular entities and the        one or more lipids;    -   d. inline mixing of the receptor targeting conjugate to the        mixture of step (c), thereby producing a mixture comprising        nanoparticles that comprise the one or more nucleic acid        molecular entities, the one or more lipids, and the receptor        targeting conjugate;    -   e. diluting the mixture of step (d) by adding a dilution buffer;        and    -   f. optionally carrying out a buffer exchange process.-   259. A pharmaceutical composition comprising nanoparticles, wherein    the nanoparticles comprise (i) one or more nucleic acid molecular    entities, (ii) one or more lipids selected from a sterol or a    derivative thereof, a phospholipid, a stealth lipid, and an amino    lipid, and (iii) a receptor targeting conjugate, wherein the    formulation is prepared by a method comprising:    -   a. providing a first solution comprising the one or more nucleic        acid molecular entities;    -   b. providing a second solution comprising (i) at least one of        the one or more lipids and (ii) at least a portion of the        receptor targeting conjugate;    -   c. mixing the first solution and the second solution, thereby        producing a mixture comprising nanoparticles;    -   d. optionally incubating the nanoparticles; and    -   e. optionally carrying out a buffer exchange process.-   260. A subject as referenced in any one of the clauses above,    wherein the subject has heterozygous familial hypercholesterolemia    (HeFH), homozygous familial hypercholesterolemia (HoFH) or clinical    atherosclerotic cardiovascular disease (ASCVD).-   261. A subject as referenced in any one of the clauses above,    wherein the subject is at high risk of cardiovascular events and    require additional lowering of low-density lipoprotein cholesterol    (LDL-C) despite maximally tolerated lipid-lowering therapy.-   262. A lipid nanoparticle composition comprising:    -   a. one or more nucleic acid molecular entities; and    -   b. a receptor targeting conjugate as referenced in any one of        the clauses above.-   263. The lipid nanoparticle composition of clause 262, wherein the    receptor targeting conjugate comprises from about 0.001 mol % to    about 20 mol % of the total lipid content.-   264. The lipid nanoparticle composition of clause 262, wherein the    receptor targeting conjugate comprises from about 0.005 mol % to    about 2 mol % of the total lipid content.-   265. The lipid nanoparticle composition of clause 262, wherein the    receptor targeting conjugate comprises from about 0.01 mol % to    about 1.0 mol % of the total lipid content present.-   266. The lipid nanoparticle composition of clause 262, wherein the    nanoparticle composition further comprising amino lipid, sterol or    its derivative thereof, phospholipid and PEG-Lipid-   267. The lipid nanoparticle composition of clause 266, wherein the    amino lipid is constituted from 1 or more amino lipids and the total    amino lipids comprises to about 10 mol % to about 70 mol % of the    total lipid content.-   268. The lipid nanoparticle composition of clause 266, wherein the    amino lipid comprises about 40 mol % to about 55 mol % of the total    lipid content.-   269. The lipid nanoparticle composition of clause 266, wherein the    amino lipid comprises about 45 mol % to about 50 mol % of the total    lipid content.-   270. The lipid nanoparticle composition of clause 266, wherein the    sterol or its derivative thereof comprises to about 10 mol % to    about 70 mol % of the total lipid content.-   271. The lipid nanoparticle of composition of clause 270, wherein    the sterol or its derivative thereof is cholesterol.-   272. The lipid nanoparticle composition of clause 266, wherein the    phospholipid comprises to about 5 mol % to about 15 mol % of the    total lipid content.-   273. The lipid nanoparticle composition of clause 266, wherein the    phospholipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).-   274. The lipid nanoparticle composition of clause 266, wherein the    stealth lipid comprises about 0 mol % to about 6 mol % of the total    lipid content present in said nanoparticle composition.-   275. The lipid nanoparticle composition of clause 266, wherein the    stealth lipid is a PEG-lipid that has a number average molecular    weight of from about 200 Da to about 5000 Da.-   276. The lipid nanoparticle composition of clause 275, wherein the    PEG-lipid has a number average molecular weight of from about 1500    Da to about 3000 Da.-   277. The lipid nanoparticle composition of clause 276, comprising:    -   a. about 0.001 mol % to about 20 mol % of the receptor targeting        conjugate of any one of the preceding clauses;    -   b. about 10 mol % to about 70 mol % of one or more amino lipids;    -   c. about 10 mol % to about 70 mol % of a sterol;    -   d. about 3 mol % to about 15 mol % of a phospholipid; and    -   e. about 0.1 mol % to about 6 mol % of a stealth lipid.-   278. The lipid nanoparticle composition of clause 277, wherein    -   a. the targeting conjugate is selected from Table 4 and        comprises 0.001 mol % to about 4 mol % of the total lipid        content;    -   b. the amino lipid comprises about 20 mol % to 70 mol % of the        total lipid content;    -   c. the sterol is cholesterol and it comprises about 20 mol % to        about 60 mol % of the total lipid content;    -   d. the phospholipid is 1,        2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and comprises        about 5 mol % to about 15 mol % of the total lipid content; and    -   e. the stealth lipid is a PEG-lipid with a number average        molecular weight of from about 200 Da to about 5000 Da and        comprises about 0 mol % to about 5 mol %.-   279. The lipid nanoparticle composition of clause 277, wherein    -   a. the targeting conjugate is selected from Table 4 and        comprises 0.001 mol % to about 2 mol % of the total lipid        content;    -   b. the amino lipid comprises about 40 mol % to 70 mol % of the        total lipid content;    -   c. the sterol is cholesterol and it comprises about 20 mol % to        about 60 mol % of the total lipid content; and    -   d. the phospholipid is 1,        2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and comprises        about 5 mol % to about 15 mol % of the total lipid content.-   280. A receptor targeting conjugate, comprising a compound of    Formula (V):

-   -   wherein,    -   a plurality of the A groups collectively comprising a receptor        targeting ligand;    -   each of L¹, L², L³, L⁴, L⁵, L⁶, L⁷, L⁸, L⁹, L¹⁰ and L¹² is        independently substituted or unsubstituted C₁-C₁₂ alkylene,        substituted or unsubstituted C₁-C₁₂ heteroalkylene, substituted        or unsubstituted C₂-C₁₂ alkenylene, substituted or unsubstituted        C₂-C₁₂ alkynylene, —(CH₂CH₂O)_(m)—, —(OCH₂CH₂)_(m)—, —O—, —S—,        —S(═O)—, —S(═O)₂—, —S(═O)(═NR¹)—, —C(═O)—, —C(═N—OR¹)—,        —C(═O)O—, —OC(═O)—, —C(═O)C(═O)—, —C(═O)NR¹—, —NR¹C(═O)—,        —OC(═O)NR¹—, —NR¹C(═O)O—, —NR¹C(═O)NR¹—, —C(═O)NR¹C(═O)—,        —S(═O)₂NR¹—, —NR¹S(═O)₂—, —NR¹—, or —N(OR¹)—;    -   L¹¹ is substituted or unsubstituted —(CH₂CH₂O)_(n)— or        substituted or unsubstituted —(OCH₂CH₂)_(n)—;    -   each R¹ is independently H or substituted or unsubstituted C₁-C₆        alkyl;    -   R comprises or is a lipid, nucleic acid, amino acid, protein, or        lipid nanoparticle;    -   m is an integer selected from 1 to 10; and    -   n is an integer selected from 1 to 200.

-   281. The receptor targeting conjugate of clause 280, wherein    -   a. the plurality of the A groups comprises a lectin receptor        targeting ligand;    -   b. each of L¹, L³, L⁴, and L⁷ comprises —(CH₂)₄—;    -   c. each of L², L⁵, and L⁸ comprises —C(═O)NH—;    -   d. each of L⁶ and L⁹ comprises —(CH₂)₃—;    -   e. L¹⁰ is —(CH₂)₁₋₃—, —CH₂CH₂O— or —CH₂O—;    -   f. L¹¹ is —(CH₂CH₂O)_(n)— or —(OCH₂CH₂)_(n)—, where n is an        integer selected from 1 to 50;    -   g. L¹² is —NH(CO)O—; and    -   h. R is selected from the group consisting of dialkylglycerol,        diacylglycerol, sterol, n-alkyl comprising C₁₀-C₃₀ carbon atoms,        branched alkyl comprising C₁₀-C₃₀ carbon atoms or tocopherol.

-   282. The receptor targeting conjugate of clause 281, wherein the n    is 1-3, 9-15, 33-39 or 41-49.

-   283. The receptor targeting conjugate of clause 281, wherein the    lectin receptor is asialoglycoprotein receptor (ASGPR) and the    plurality of the A groups comprise N-acetylgalactosamine, galactose    or combination thereof

-   284. The receptor targeting conjugate of clause 280, wherein each of    the A groups is N-acetylgalactosamine

-   285. The receptor targeting conjugate of clause 280, wherein the    receptor targeting conjugate is a conjugate selected from 1001-1019,    1060, 1065, 1066 and 1075-1085 in Table 4.-   286. The receptor targeting conjugate of clause 280, wherein the    receptor targeting conjugate is

-   287. The receptor targeting conjugate of clause 280, wherein the R    comprises one or more of fatty alcohols, fatty acids, glycerolipids,    glycerophospholipids, sphingolipids, saccharolipids, polyketides, or    sterols or derivatives thereof.-   288. The receptor targeting conjugate of clause 280, wherein the R    is a lipid nanoparticle that comprises one or more mRNA encoding one    or more gene editor nuclease(s) or base editors and one or more    guide RNAs.-   289. A method for reducing the risk of coronary disease in a subject    in need thereof, the method comprising administering to the subject    a lipid nanoparticle encapsulating a payload comprising one or more    pharmaceutically active agents, wherein the lipid nanoparticle    further comprises a receptor targeting conjugate of Formula (V):

-   -   wherein,    -   a plurality of the A groups collectively comprising a receptor        targeting ligand;    -   each of L¹, L², L³, L⁴, L⁵, L⁶, L⁷, L⁸, L⁹, L¹⁰ and L¹² is        independently substituted or unsubstituted C₁-C₁₂ alkylene,        substituted or unsubstituted C₁-C₁₂ heteroalkylene, substituted        or unsubstituted C₂-C₁₂ alkenylene, substituted or unsubstituted        C₂-C₁₂ alkynylene, —(CH₂CH₂O)_(m)—, —(OCH₂CH₂)_(m)—, —O—, —S—,        —S(═O)—, —S(═O)₂—, —S(═O)(═NR¹)—, —C(═O)—, —C(═N—OR¹)—,        —C(═O)O—, —OC(═O)—, —C(═O)C(═O)—, —C(═O)NR¹—, —NR¹C(═O)—,        —OC(═O)NR¹—, —NR¹C(═O)O—, —NR¹C(═O)NR¹—, —C(═O)NR¹C(═O)—,        —S(═O)₂NR¹—, —NR¹S(═O)₂—, —NR¹—, or —N(OR¹)—;    -   L¹¹ is substituted or unsubstituted —(CH₂CH₂O)_(n)— or        substituted or unsubstituted —(OCH₂CH₂)_(n)—;    -   each R¹ is independently H or substituted or unsubstituted C₁-C₆        alkyl;    -   R comprises or is a lipid;    -   m is an integer selected from 1 to 10; and    -   n is an integer selected from 1 to 200.

-   290. The method of clause 289, wherein    -   a. the plurality of A groups comprises a lectin receptor        targeting ligand;    -   b. each of L¹, L³, L⁴, and L⁷ comprises —(CH₂)₄—;    -   c. each of L², L⁵, and L⁸ comprises —C(═O)NH—;    -   d. each of L⁶ and L⁹ comprises —(CH₂)₃—;    -   e. L¹⁰ is —(CH₂)₁₋₃—, —CH₂CH₂O— or —CH₂O—;    -   f. L¹¹ is —(CH₂CH₂O)_(n)— or —(OCH₂CH₂)_(n)—, where n is an        integer selected from 1 to 50;    -   g. L¹² is —NH(CO)O—; and    -   h. R is selected from the group consisting of dialkylglycerol,        diacylglycerol, sterol, n-alkyl comprising C₁₀-C₃₀ carbon atoms,        branched alkyl comprising C₁₀-C₃₀ carbon atoms or tocopherol.

-   291. The method of clause 289, wherein the receptor targeting    conjugate is a conjugate selected from 1001-1019, 1060, 1065, 1066    and 1075-1085 in Table 4.

-   292. The method of clause 289, wherein the receptor targeting    conjugate is conjugate 1004 in Table 4.

-   293. The method of clause 292, wherein the lipid nanoparticle    comprising the receptor targeting conjugate provides an improved    delivery in LDLr deficient mammal as determined by percent editing    of at least 50% higher than a corresponding lipid nanoparticle    without the receptor targeting conjugate.

-   294. The method of clause 292, wherein the lipid nanoparticle    comprising the receptor targeting conjugate provides an improved    delivery in a mammal that lacks ApoE as determined by percent    editing of at least 50% higher than a corresponding lipid    nanoparticle without the receptor targeting conjugate.

-   295. The method of clause 289, wherein the one or more active agents    comprise an mRNA encoding a gene editor nuclease or a base editor    and one or more guide RNAs.

-   296. The method of clause 295, wherein the mRNA is an adenosine base    editor and the one or more guide RNAs are complementary to (i) a    segment of PCSK9 gene, (ii) a segment of ANGPTL3 gene, or both (i)    and (ii).

-   297. The method of clause 295, wherein at least one of the one or    more guide RNAs is selected from guide RNA sequences of SEQ ID NOs:    121-126 of Table 5.

-   298. The method of clause 289, wherein the receptor targeting    conjugate comprises from about 0.001 mol % to about 0.5 mol % of the    total excipients in the lipid nanoparticle.

-   299. A method of preparing a formulation comprising GalNAc-lipid    nanoparticles, wherein the nanoparticles comprise (i) one or more    nucleic acid active agents, (ii) lipid excipients comprising sterol    or a derivative thereof, a phospholipid, a stealth lipid, and an    amino lipid, and (iii) a GalNAc-lipid receptor targeting conjugate,    the method comprising:    -   a. providing a first solution comprising the one or more nucleic        acid active agents in aqueous buffer;    -   b. providing a second solution comprising (i) the lipid        excipient and (ii) at least a portion of the receptor targeting        conjugate in a water-miscible organic solvent such as ethanol;    -   c. mixing the first solution and the second solution;    -   d. incubating a mixture of the first and second solutions to        form GalNAc-lipid nanoparticles; and    -   e. optionally carrying out one or more dilution, buffer        exchange, concentration, filtration, and GalNAc-lipid        nanoparticle evaluation processes.

-   300. The method of clause 299, wherein the GalNAc-lipid receptor    targeting conjugate is selected from the structures identified in    Table 4.

-   301. The method of clause 299, wherein the mixing is performed by an    inline mixing apparatus having a first mixing chamber that includes    a first port that separately introduces the first solution to the    first mixing chamber and a second port that separately and    simultaneously introduces the second solution into the first mixing    chamber.

-   302. The method of clause 301, further comprising adding a second    portion of the receptor targeting conjugate after the first solution    and the second solution are mixed, wherein the addition of the    second portion of the receptor targeting conjugate is pre-dissolved    in a water miscible organic solvent and combined with an aqueous    solution to form an aqueous dilution buffer that is mixed with the    previously mixed first and second solutions in a second mixing    chamber conjoined with and downstream from the first mixing chamber    of the inline mixing apparatus prior to incubation.

-   303. The method of clause 302, further comprising a buffer exchange    process after the second portion of the receptor targeting conjugate    is added.

-   304. A method of preparing a formulation comprising GalNAc-lipid    nanoparticles, wherein the nanoparticles comprise (i) one or more    nucleic acid molecular entities, (ii) lipid excipient comprising one    or more sterols or a derivative thereof, a phospholipids, a stealth    lipids, or an amino lipids, and (iii) one or more GalNAc-lipid    receptor targeting conjugates, the method comprising:    -   a. providing a first solution comprising the one or more nucleic        acid molecular entities in an aqueous buffer;    -   b. providing a second solution comprising at least one of the        one or more lipid excipients in a water-miscible organic        solvent;    -   c. providing a third solution comprising at least a portion of        the receptor targeting conjugate;    -   d. mixing the first solution, the second solution, and the third        solution in one or more mixing chambers wherein each solution is        introduced separately via an inlet port to the one or more        mixing chambers;    -   e. incubating a mixture of the first, second and third solutions        to form GalNAc-lipid nanoparticles; and    -   f. optionally carrying out one or more dilution, a buffer        exchange, concentration, filtration, and GalNAc-lipid        nanoparticle evaluation processes.

-   305. The method of clause 304, wherein the first solution comprises    an aqueous buffer, and wherein the second solution and the third    solution are each independently prepared from a water-miscible    alcohol.

-   306. The method of clause 304, wherein the first solution, the    second solution, and the third solution are introduced to a mixer    simultaneously.

-   307. The method of clause 304, wherein the first solution, the    second solution, and the third solution are introduced to a mixer    sequentially prior to incubation.

-   308. The method of clause 304, wherein the first solution, the    second solution, and the third solution are combined in an in-line    mixer apparatus having a first mixing chamber conjoined to a second    downstream mixing chamber, wherein the first and second solutions    are pre-mixed in the first mixing chamber and immediately flow into    a second mixing chamber and wherein the third solution is mixed with    the first and second solution in the second mixing chamber; and    wherein the third solution may optionally comprise a targeting    conjugate and/or lipid excipients pre-dissolved in a water-miscible    organic solvent mixed in or diluted with an aqueous dilution buffer.

-   309. The method of clause 304, wherein the water-miscible organic    solvent is ethanol.

-   310. A targeting moiety or receptor targeting conjugate as    referenced in any one of the foregoing clauses, wherein the    targeting moiety or receptor targeting conjugate has a structure    represented in Table 1.

-   311. A GalNAc conjugate as referenced in any one of the foregoing    clauses, wherein the GalNAc conjugate comprises a sequence shown in    Table 2 or Table 3.

-   312. A receptor targeting conjugate as referenced in any one of the    foregoing clauses, wherein the receptor targeting conjugate    comprises a structure of Table 4.

-   313. A guide RNA as referenced in any one of the foregoing clauses,    wherein the guide RNA comprises a sequence shown in Table 5.

-   314. A lipid excipient as referenced in any one of the foregoing    clauses, wherein the lipid excipient comprises a structure of Table    6 or Table 7.

-   315. A lipid nanoparticle as referenced in any one of the foregoing    clauses, wherein the lipid nanoparticle comprises a composition of    Table 8, Table 9, Table 10, Table 11, Table 12, or Table 13.

It will also be appreciated from reviewing the present disclosure, thatit is contemplated that the one or more aspects or features presented inone set of clauses may also be included in other clauses or incombination with the one or more aspects or features in other clauses.

1.-9. (canceled)
 10. A method for reducing the risk of coronary diseasethrough the reduction of LDL cholesterol and/or triglyceride levels in asubject, the method comprising administering to the subject a lipidnanoparticle encapsulating a nucleic acid payload wherein, upon deliveryof the lipid nanoparticle into the subject's cells, the payload isengineered to generate a gene editor that is capable of modifying one ormore genes involved in controlling or regulating LDL and/or triglyceridelevels in the subject, wherein the lipid nanoparticle further comprisesa compound of Formula (V):

wherein, a plurality of the A groups collectively comprising a receptortargeting ligand; each of L¹, L², L³, L⁴, L⁵, L⁶, L⁷, L⁸, L⁹, L¹⁰ andL¹² is independently substituted or unsubstituted C₁-C₁₂ alkylene,substituted or unsubstituted C₁-C₁₂ heteroalkylene, substituted orunsubstituted C₂-C₁₂ alkenylene, substituted or unsubstituted C₂-C₁₂alkynylene, —(CH₂CH₂O)_(m)—, —(OCH₂CH₂)_(m)—, —O—, —S—, —S(═O)—,—S(═O)₂—, —S(═O)(═NR¹)—, —C(═O)—, —C(═N—OR¹)—, —C(═O)O—, —OC(═O)—,—C(═O)C(═O)—, —C(═O)NR¹—, —NR¹C(═O)—, —OC(═O)NR¹—, —NR¹C(═O)O—,—NR¹C(═O)NR¹—, —C(═O)NR¹C(═O)—, —S(═O)₂NR¹—, —NR¹S(═O)₂—, —NR¹—, or—N(OR¹)—; L¹¹ is substituted or unsubstituted —(CH₂CH₂O)_(n)— orsubstituted or unsubstituted —(OCH₂CH₂)_(n)—; each R¹ is independently Hor substituted or unsubstituted C₁-C₆ alkyl; R comprises a lipid,nucleic acid, amino acid, or protein; m is an integer selected from 1 to10; and n is an integer selected from 1 to 200, wherein each of the Agroups is a N-acetylgalactosamine moiety


11. The method of claim 10, wherein a. each of L¹, L³, L⁴, and L⁷comprises —(CH₂)₄—; b. each of L², L⁵, and L⁸ comprises —C(═O)NH—; c.each of L⁶ and L⁹ comprises —(CH₂)₃—; d. L¹⁰ is —(CH₂)₁₋₃—, —CH₂CH₂O— or—CH₂O—; e. L¹¹ is —(CH₂CH₂O)_(n)— or —(OCH₂CH₂)_(n)—, where n is aninteger selected from 1 to 50; f. L¹² is —NH(CO)O—, —NHC(═O)—, or—OC(═O)NH—; and g. R is selected from the group consisting of dialkylglycerol, diacyl glycerol, sterol, n-alkyl comprising C₁₀-C₃₀ carbonatoms, branched alkyl comprising C₁₀-C₃₀ carbon atoms or tocopherol. 12.The method of claim 10, wherein the compound is a compound selected fromthe following:

wherein each of the p and q is independently an integer from 1 to 5, andn is an integer from 1 to 50,

wherein n is an integer from 1 to 50;

wherein each of the p and q is independently an integer from 1 to 5, andn is an integer from 1 to 50,

wherein n is an integer from 1 to 50;

wherein each of the p and q is independently an integer from 1 to 5, andn is an integer from 1 to 50,

wherein each of the p and q is independently an integer from 1 to 5, andn is an integer from 1 to 50,

wherein each of the p and q is independently an integer from 1 to 5, andn is an integer from 1 to 50,

wherein n is an integer from 1-50;


13. The method of claim 10, wherein the compound is compound


14. The method of claim 13, wherein the lipid nanoparticle comprisingthe compound provides an improved liver delivery in a LDLr deficientmammal as determined by percent editing of at least 50% higher than acorresponding lipid nanoparticle without the compound.
 15. The method ofclaim 13, wherein the lipid nanoparticle comprising the compoundprovides an improved liver delivery in a mammal that lacks ApoE asdetermined by percent editing of at least 50% higher than acorresponding lipid nanoparticle without the compound.
 16. The method ofclaim 10, wherein the nucleic acid payload comprises an mRNA encoding agene editor nuclease or a base editor and one or more guide RNAs. 17.The method of claim 16, wherein the mRNA is an adenosine base editor andthe one or more guide RNAs are complementary to (i) a segment of PCSK9gene, (ii) a segment of ANGPTL3 gene, or both (i) and (ii).
 18. Themethod of claim 16, wherein at least one of the one or more guide RNAsis selected from guide RNA sequences of SEQ ID NOs: 121-126 of Table 5.19. The method of claim 10, wherein the compound comprises from about0.001 mol % to about 1.0 mol % of the total lipid nanoparticleexcipients. 20.-30. (canceled)
 31. The method of claim 10, wherein thecompound comprises from about 0.01 mol % to about 0.1 mol % of totallipid nanoparticle excipients.
 32. The method of claim 10, wherein thecompound comprises from about 0.03 mol % to about 0.07 mol % of totallipid nanoparticle excipients.
 33. The method of claim 10, wherein thecompound comprises from about 0.05 mol % of total lipid nanoparticleexcipients.
 34. The method of claim 10, wherein n is 1-3, 9-15, 33-39,or 41-49.
 35. The method of claim 10, wherein the compound is

wherein each of the p and q is independently an integer from 1 to 5, andn is an integer from 33 to
 39. 36. The method of claim 35, wherein eachof the p and q is
 2. 37. The method of claim 35, wherein n is
 35. 38.The method of claim 10, wherein R comprises one or more of fattyalcohols, fatty acids, glycerolipids, glycerophospholipids,sphingolipids, saccharolipids, polyketides, or sterols or derivatesthereof.
 39. The method of claim 10, wherein the lipid nanoparticlefurther comprises one or more mRNA encoding one or more gene editornuclease(s) or base editors and one or more guide RNAs.
 40. The methodof claim 10, wherein L¹, L⁴, and L⁷ is independently substituted orunsubstituted C₁-C₁₂ alkylene.
 41. The method of claim 40, wherein L¹,L⁴, and L⁷ is independently substituted or unsubstituted C₂-C₆ alkylene.42. The method of claim 41, wherein L¹, L⁴, and L⁷ is C₄ alkylene. 43.The method of claim 10, wherein each L², L⁵, and L⁸ is independently—C(═O)N(R¹)—, —N(R¹)C(═O)—, —OC(═O)N(R¹)—, —N(R¹)C(═O)O—, or—N(R¹)C(═O)N(R¹).
 44. The method of claim 10, wherein A binds to alectin.
 45. The method of claim 10, wherein R is dialkyl glycerol.
 46. Acomposition comprising: (i) one or more lipids selected from a sterol orderivative thereof, a phospholipid, a stealth lipid, and an amino lipid,and (ii) a compound of Formula (V):

wherein, a plurality of the A groups collectively comprising a receptortargeting ligand; each of L¹, L², L³, L⁴, L⁵, L⁶, L⁷, L⁸, L⁹, L¹⁰ andL¹² is independently substituted or unsubstituted C₁-C₁₂ alkylene,substituted or unsubstituted C₁-C₁₂ heteroalkylene, substituted orunsubstituted C₂-C₁₂ alkenylene, substituted or unsubstituted C₂-C₁₂alkynylene, —(CH₂CH₂O)_(m)—, —(OCH₂CH₂)_(m)—, —O—, —S—, —S(═O)—,—S(═O)₂—, —S(═O)(═NR¹)—, —C(═O)—, —C(═N—OR¹)—, —C(═O)O—, —OC(═O)—,—C(═O)C(═O)—, —C(═O)NR¹—, —NR¹C(═O)—, —OC(═O)NR¹—, —NR¹C(═O)O—,—NR¹C(═O)NR¹—, —S(═O)₂NR¹—, —NR¹S(═O)₂—, —NR¹—, or —N(OR¹)—; L¹¹ issubstituted or unsubstituted —(CH₂CH₂O)_(n)— or substituted orunsubstituted —(OCH₂CH₂)_(n)—; each R¹ is independently H or substitutedor unsubstituted C₁-C₆ alkyl; R comprises a lipid, nucleic acid, aminoacid, protein, or lipid nanoparticle; m is an integer selected from 1 to10; and n is an integer selected from 1 to 200, wherein each of the Agroups is a N-acetylgalactosamine moiety


47. The composition of claim 46, wherein a. each of L¹, L³, L⁴, and L⁷comprises —(CH₂)₄—; b. each of L², L⁵, and L⁸ comprises —C(═O)NH—; c.each of L⁶ and L⁹ comprises —(CH₂)₃—; d. L¹⁰ is —(CH₂)₁₋₃—, —CH₂CH₂O— or—CH₂O—; e. L¹¹ is —(CH₂CH₂O)_(n)— or —(OCH₂CH₂)_(n)—, where n is aninteger selected from 1 to 50; f. L¹² is —NH(CO)O— or —NHC(═O)—; and g.R is selected from the group consisting of dialkylglycerolyl,diacylglycerolyl, sterol, n-alkyl comprising C₁₀-C₃₀ carbon atoms,branched alkyl comprising C₁₀-C₃₀ carbon atoms or tocopherol.
 48. Thecomposition of claim 46, wherein the n is 1-3, 9-15, 33-39, or 41-49.49. The composition of claim 46, wherein the compound is a compoundselected from the following:

wherein each of the p and q is independently an integer from 1 to 5, andn is an integer from 1 to 50;

wherein n is an integer from 1 to 50;

wherein each of the p and q is independently an integer from 1 to 5, andn is an integer from 1 to 50;

wherein n is an integer from 1 to 50;

wherein each of the p and q is independently an integer from 1 to 5, andn is an integer from 1 to 50;

wherein each of the p and q is independently an integer from 1 to 5, andn is an integer from 1 to 50;

wherein each of the p and q is independently an integer from 1 to 5, andn is an integer from 1 to 50;

wherein n is an integer from 1-50;


50. The composition of claim 46, wherein the compound is

wherein each of the p and q is independently an integer from 1 to 5, andn is an integer from 33 to
 39. 51. The composition of claim 50, whereineach p and q are
 2. 52. The composition of claim 50, wherein n is 35.53. The composition of claim 46, wherein the compound is


54. The composition of claim 50, where the composition further comprisesa plurality of compounds where n is an integer from 33 to
 39. 55. Thecomposition of claim 46, wherein the R comprises one or more of fattyalcohols, fatty acids, glycerolipids, glycerophospholipids,sphingolipids, saccharolipids, polyketides, or sterols or derivativesthereof.
 56. The composition of claim 46, wherein the compositionfurther comprises one or more mRNA encoding one or more gene editornuclease(s) or base editors and one or more guide RNAs.
 57. Thecomposition of claim 46, wherein L¹, L⁴, and L⁷ is independentlysubstituted or unsubstituted C₁-C₁₂ alkylene.
 58. The composition ofclaim 57, wherein L¹, L⁴, and L⁷ is independently substituted orunsubstituted C₂-C₆ alkylene.
 59. The composition of claim 58, whereinL¹, L⁴, and L⁷ is C₄ alkylene.
 60. The composition of claim 46, whereineach L², L⁵, and L⁸ is independently —C(═O)N(R¹)—, —N(R¹)C(═O)—,—OC(═O)N(R¹)—, —N(R¹)C(═O)O—, or —N(R¹)C(═O)N(R¹).
 61. The compositionof claim 46, wherein A binds to a lectin.
 62. The composition of claim46, wherein R is dialkyl glycerolyl.
 63. The composition of claim 46,wherein the compound of Formula (V) comprises from about 0.001 mol % toabout 1.0 mol % of total excipient composition.
 64. The composition ofclaim 46, wherein the compound of Formula (V) comprises from about 0.05%of total excipient composition.
 65. The composition of claim 46, furthercomprising one or more nucleic acid molecular entities.
 66. Thecomposition of claim 65, wherein the one or more nucleic acid molecularentities is a nuclease mRNA.
 67. The composition of claim 65, whereinthe one or more nucleic acid molecular entities is a guide RNA.
 68. Thecomposition of claim 65, wherein the one or more nucleic acid molecularentities comprises one or more nuclease mRNA and one or more guide RNA.69. The composition of claim 68, wherein the guide RNA comprises asingle guide RNA or a dual guide RNA.
 70. The composition of claim 69,wherein the guide RNA targets a gene associated with coronary disease.71. The composition of claim 68, wherein the guide RNA targets PCSK9 orANGPTL3.
 72. The composition of claim 66, wherein the nuclease mRNAencodes a nickase.
 73. The composition of claim 72, wherein the mRNAencodes a base editor.
 74. The composition of claim 46, wherein thecomposition is a pharmaceutical composition.
 75. The method of claim 10,wherein the effect of modifying one or more genes comprises aninsertion, deletion, substitution, alteration of a nucleotide in anucleic acid sequence, or a combination thereof.
 76. A compound ofFormula (V):

wherein, a plurality of the A groups collectively comprising a receptortargeting ligand; each of L¹, L², L³, L⁴, L⁵, L⁶, L⁷, L⁸, L⁹, L¹⁰ andL¹² is independently substituted or unsubstituted C₁-C₁₂ alkylene,substituted or unsubstituted C₁-C₁₂ heteroalkylene, substituted orunsubstituted C₂-C₁₂ alkenylene, substituted or unsubstituted C₂-C₁₂alkynylene, —(CH₂CH₂O)_(m)—, —(OCH₂CH₂)_(m)—, —O—, —S—, —S(═O)—,—S(═O)₂—, —S(═O)(═NR¹)—, —C(═O)—, —C(═N—OR¹)—, —C(═O)O—, —OC(═O)—,—C(═O)C(═O)—, —C(═O)NR¹—, —NR¹C(═O)—, —OC(═O)NR¹—, —NR¹C(═O)O—,—NR¹C(═O)NR¹—, —S(═O)₂NR¹—, —NR¹S(═O)₂—, —NR¹—, or —N(OR¹)—; L¹¹ issubstituted or unsubstituted —(CH₂CH₂O)_(n)— or substituted orunsubstituted —(OCH₂CH₂)_(n)—; each R¹ is independently H or substitutedor unsubstituted C₁-C₆ alkyl; R comprises a lipid, nucleic acid, aminoacid, protein, or lipid nanoparticle; m is an integer selected from 1 to10; and n is an integer selected from 1 to 200, wherein each of the Agroups is a N-acetylgalactosamine moiety


77. The compound of claim 76, wherein a. each of L¹, L³, L⁴, and L⁷comprises —(CH₂)₄—; b. each of L², L⁵, and L⁸ comprises —C(═O)NH—; c.each of L⁶ and L⁹ comprises —(CH₂)₃—; d. L¹⁰ is —(CH₂)₁₋₃—, —CH₂CH₂O— or—CH₂O—; e. L¹¹ is —(CH₂CH₂O)_(n)— or —(OCH₂CH₂)_(n)—, where n is aninteger selected from 1 to 50; f. L¹² is —NH(CO)O— or —NHC(═O)—; and g.R is selected from the group consisting of dialkylglycerolyl,diacylglycerolyl, sterol, n-alkyl comprising C₁₀-C₃₀ carbon atoms,branched alkyl comprising C₁₀-C₃₀ carbon atoms or tocopherol.
 78. Thecompound of claim 76, wherein the n is 1-3, 9-15, 33-39, or 41-49. 79.The compound of claim 76, wherein the compound is a compound selectedfrom the following:

wherein each of the p and q is independently an integer from 1 to 5, andn is an integer from 1 to 50;

wherein n is an integer from 1 to 50;

wherein each of the p and q is independently an integer from 1 to 5, andn is an integer from 1 to 50;

wherein n is an integer from 1 to 50;

wherein each of the p and q is independently an integer from 1 to 5, andn is an integer from 1 to 50;

wherein each of the p and q is independently an integer from 1 to 5, andn is an integer from 1 to 50;

wherein each of the p and q is independently an integer from 1 to 5, andn is an integer from 1 to 50;

wherein n is an integer from 1-50;


80. The compound of claim 76, wherein the compound is

wherein each of the p and q is independently an integer from 1 to 5, andn is an integer from 33 to
 39. 81. The compound of claim 80, whereineach p and q are
 2. 82. The compound of claim 80, wherein n is
 35. 83.The compound of claim 76, wherein the compound is


84. The compound of claim 80, where the pharmaceutical compositionfurther comprises a plurality of compounds where n is an integer from 33to
 39. 85. The compound of claim 76, wherein the R comprises one or moreof fatty alcohols, fatty acids, glycerolipids, glycerophospholipids,sphingolipids, saccharolipids, polyketides, or sterols or derivativesthereof.